Dorzolamide-induced relaxation of porcine retinal arterioles in vitro depends on nitric oxide but not on acidosis in vascular smooth muscle cells.

The carbonic anhydrase inhibitor dorzolamide can induce relaxation of retinal arterioles with a consequent increase in blood flow and oxygenation of the retina. It has been shown that the mechanisms underlying this relaxation are independent of extracellular acidosis and CO2. The purpose of the present study was to investigate the possible involvement of nitric oxide (NO) and intracellular acidosis in dorzolamide-induced relaxation of retinal arterioles. Porcine retinal arterioles were mounted in a wire myograph and dorzolamide induced relaxation was studied after 1) the addition of the NO synthase inhibitor l-NAME (3 × 10(-4) M) or the guanylyl cyclase inhibitor ODQ (3 × 10(-6) M), and 2) after loading the smooth muscle cells with the pH sensitive fluorophore SNARF-1-AM and studying changes in vascular tone and intracellular fluorescence after the induction of hypoxia, addition of lactate (10(-2) M), and extracellular acidification (pH = 7.0) alone and in the presence of dorzolamide (10(-3) M). Dorzolamide significantly relaxed retinal arterioles (p < 0.03), and the effect was significantly higher in the presence of perivascular tissue than in isolated vessels at the highest concentration (p < 0.01). In the presence of perivascular tissue dorzolamide-induced relaxation could be reduced by NO inhibition (p < 0.02). Dorzolamide increased intracellular acidification (p < 0.02) during extracellular acidosis, but there was no relation between relaxation and intracellular acidosis. In conclusion, dorzolamide-induced vasorelaxation depends on NO and the perivascular retinal tissue, but is independent of acidification in the extracellular and the intracellular space of retinal vascular smooth muscle cells. Other factors than NO and acidification are involved in dorzolamide-induced relaxation of retinal arterioles.

The viability of cells obtained using the Reamer-Irrigator-Aspirator system and in bone graft from the iliac crest.

We hypothesised that cells obtained via a Reamer-Irrigator-Aspirator (RIA) system retain substantial osteogenic potential and are at least equivalent to graft harvested from the iliac crest. Graft was harvested using the RIA in 25 patients (mean age 37.6 years (18 to 68)) and from the iliac crest in 21 patients (mean age 44.6 years (24 to 78)), after which ≥ 1 g of bony particulate graft material was processed from each. Initial cell viability was assessed using Trypan blue exclusion, and initial fluorescence-activated cell sorting (FACS) analysis for cell lineage was performed. After culturing the cells, repeat FACS analysis for cell lineage was performed and enzyme-linked immunosorbent assay (ELISA) for osteocalcin, and Alizarin red staining to determine osteogenic potential. Cells obtained via RIA or from the iliac crest were viable and matured into mesenchymal stem cells, as shown by staining for the specific mesenchymal antigens CD90 and CD105. For samples from both RIA and the iliac crest there was a statistically significant increase in bone production (both p < 0.001), as demonstrated by osteocalcin production after induction. Medullary autograft cells harvested using RIA are viable and osteogenic. Cell viability and osteogenic potential were similar between bone grafts obtained from both the RIA system and the iliac crest.

Openers of small conductance calcium-activated potassium channels selectively enhance NO-mediated bradykinin vasodilatation in porcine retinal arterioles.

BACKGROUND AND PURPOSE: Small (SK(Ca) or K(Ca)2) and intermediate (IK(Ca) or K(Ca)3.1) conductance calcium-activated potassium channels are involved in regulation of vascular tone and blood pressure. The present study investigated whether NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) and CyPPA (cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine), which are selective openers of SK(Ca) and IK(Ca) channels and of SK(Ca)2 and SK(Ca)3 channels, respectively, enhance endothelium-dependent vasodilatation in porcine retinal arterioles. EXPERIMENTAL APPROACH: In porcine retinal arterioles, SK(Ca)3 and IK(Ca) protein localization was examined by immunolabelling. Endothelial cell calcium was measured by fluorescence imaging. For functional studies, arterioles with internal diameters of 116 +/- 2 microm (n = 276) were mounted in microvascular myographs for isometric tension recordings. KEY RESULTS: SK(Ca)3 and IK(Ca) protein was localized in the endothelium. Bradykinin, but not NS309 or CyPPA increased endothelial cell calcium. Pre-incubation with NS309 or CyPPA enhanced bradykinin relaxation without changing endothelial cell calcium. This enhanced relaxation was abolished by blocking SK(Ca) channels with apamin. In the presence of NS309 or CyPPA, mainly inhibition of NO synthase with asymmetric dimethylarginine, but also inhibition of cyclooxygenase with indomethacin, reduced bradykinin relaxation. Bradykinin relaxation was completely abolished by NO synthase and cyclooxygenase inhibition together with a NO scavenger, oxyhaemoglobin. CONCLUSIONS AND IMPLICATIONS: In porcine retinal arterioles, bradykinin increases endothelial cell calcium leading to activation of SK(Ca) and IK(Ca) channels. Without altering endothelial cell calcium, NS309 and CyPPA open SK(Ca) channels that enhance NO-mediated bradykinin relaxations. These results imply that opening SK(Ca) channels improves endothelium-dependent relaxation and makes this channel a potential target for treatments aimed at restoring retinal blood flow.

Effects of feeding fumonisin B1 in lactating sows and their suckling pigs.

The mycotoxin fumonisin B1 (FB1) produced by Fusarium moniliforme in corn causes pulmonary edema in finishing swine. Effects of lower nonlethal amounts and effects in lactating sows with suckling pigs are unknown. An initial study was conducted to determine a nonlethal concentration of FB1 for lactating sows; whether ingested FB1 could be detected in the milk; and whether toxicosis could be detected in the pigs, as determined by necropsy. Another study was conducted to determine toxicosis in the pigs by measuring liver sphinganine-to-sphingosine ratio, and whether ingested FB1 affected T-lymphocyte function in sows and their pigs. Furthermore, sows of this study were maintained in controlled hot (27 to 32 C, 50 to 70% relative humidity) and thermoneutral (21 C, 55% relative humidity) environments to determine whether high temperature exacerbated the effects of FB1. In the first study, 100 micrograms of FB1/g of corn soybean meal diet was found to be nonlethal when fed for 14 days. Fumonisin B1 was not detected in the milk at 30 ppb and lesions were not found in the necropsied pigs, including 1 from a sow that died of porcine pulmonary edema syndrome after ingesting FB1 at a concentration of 175 ppm. In the second study, differences in liver sphinganine-to-sphingosine ratio of pigs were not found. Expressions of cell surface antigens on blood lymphocytes and lymphocyte proliferation response to various mitogens were not affected by FB1 or high temperature in sows or their pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimelanoma antibodies in swine with spontaneously regressing melanoma.

Sinclair swine provide a unique model for studying mechanisms of tumor regression because they are born with melanomas that spontaneously regress approximately 10 weeks after birth. To examine whether an antitumor immune response is present in these animals, and, if so, to study its relation to tumor regression, 38 sera specimens collected at different times from 13 swine born with melanomas were tested for melanoma antibodies by immunoprecipitation and SDS-PAGE analysis of 125I labelled swine melanoma macromolecules. Antibodies to melanoma were present in 13 (100%) of the swine versus 1 of 3 control swine. The antibodies were directed to antigens of approximately 45, 68-75, or 100 kDa. These antigens were also expressed on human melanomas and normal melanocytes but on only one of five unrelated tumors. The incidence and level of these antibodies increased with time. Antibodies to the 45, 68-75, and 100 kDa antigens were present in 36%, 55%, and 9%, respectively, of sera collected prior to 7 weeks of age, but in 80%, 100%, and 37% of sera collected between 7 and 20 weeks (P < 0.05). The rise in melanoma antibodies usually preceded or appeared together with tumor regression and loss of pigmentation. These findings indicate that Sinclair swine with melanomas have antibodies to antigens preferentially expressed on pigment cells, and support the hypothesis that the regression phenomenon and the vitiligo-like skin depigmentation result from immune responses to common antigens shared by normal and malignant swine pigment cells.

GM-CSF is required for the Pseudomonas exotoxin A-induced proliferation of immature T cells in athymic mice.

Previous studies have demonstrated that Pseudomonas exotoxin A stimulated the proliferation of immature T lymphocytes within the splenocytes of athymic mice. These studies were performed to determine which lymphokines were involved in the proliferation of the immature T cells. The results of this study indicate that exotoxin A does not induce the production of interleukin-2 or tumor necrosis factor from B cell-depleted splenotypes from athymic mice. However, exotoxin A does induce the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from B cell-depleted splenocytes. Furthermore, the GM-CSF was shown to be produced by a Thy1+, CD4-, CD8- T lymphocyte. The addition of anti-GM-CSF antibody abrogates the exotoxin A-induced proliferation of B cell-depleted splenocytes from athymic mice. Thus, these data indicate that exotoxin A induces the production of GM-CSF from immature T lymphocytes within the splenocytes of athymic mice and the exotoxin A-induced proliferation of these immature T cells is dependent on the presence of GM-CSF.

Analyses of monoclonal antibodies reactive with porcine CD44 and CD45.

Twenty-six monoclonal antibodies (mAbs), assigned to the CD44/CD45 section of the First International Swine CD Workshop, were compared for their reactivity against a selected group of target cells by one- and two-color flow cytometric analysis. Based on staining and reactivity patterns the 26 mAbs were assigned to six groups, group F1 mAbs were designated CD44 mAbs; and groups F2 and F3 as CD45 mAbs. With the information available, a CD designation could not be given to the mAbs in groups F4, F5 or F6 consisting of four, three and four mAbs each, respectively. The reactivity of all six mAbs in group F1 (MAC35, 25-32, PORC24A, H22A, BAG40A, and BAT31A) was blocked by soluble porcine CD44. One mAb in this group (MAC325) reacted with a cell surface protein with a molecular weight of 80 kDa and was designated as CD44; the other five mAbs were designated as wCD44 because no molecular weight was known. Blocking experiments utilizing a cross reactive anti-human CD44 (mAb Z062) allowed the definition of the wCD44a epitope recognized by mAbs PORC24A and H22A. The group F2 mAbs (74-9-3; MAC323; K252.1E4; and 2A5) were designated as CD45 based on their broad reactivity pattern with lymphoid and myeloid cells and their ability to immunoprecipitate three polypeptides with an apparent molecular weight of 226, 210 and 190 kDa. The F3 mAbs (MAC327; MAC326; 3a56 and -a2) were designated as CD45R based on their restricted reactivity against lymphoid and myeloid target cells, and their ability to immunoprecipitate either two polypeptides with an apparent molecular weight of 226 and 210 kDa (mAbs MAC327 and MAC326) or a single polypeptide with an apparent molecular weight of 210 kDa (mAbs-a2 and 3a56). Sequential immunoprecipitation analyses confirmed the relatedness of the F2 and F3 group mAbs. The work conducted for this first workshop led to the definition of six mAbs specific for CD44, four mAbs specific for CD45, and four mAbs specific for CD45R which should prove to be very valuable reagents for the study of the porcine immune system.

Proliferation of immature T cells within the splenocytes of athymic mice by Pseudomonas exotoxin A.

Pseudomonas aeruginosa exotoxin A has been shown to stimulate splenocytes from athymic nude mice. The present studies were performed to characterize the exotoxin A-responsive cells within the splenocytes of athymic nude mice. The results of these studies indicate that exotoxin A-responsive cells were represented in the nylon wool-adherent cell population and in the Ig-depleted splenocyte population (IgNA). Flow microfluorimetry analysis indicated that exotoxin A induced the expansion of Thy1+, CD3-, CD4-, and CD8- cells which also expressed the heat-stable antigen (HSA), an antigen expressed on immature T lymphocytes. Depletion of Thy1+ cells abrogated the exotoxin A-induced proliferation; however, depletion of CD4+ and CD8+ cells did not affect the exotoxin A-induced proliferation of IgNA cells. Thus, the results indicate that exotoxin A stimulates the proliferation of immature T cells within the splenocytes of nude mice that are Thy1+, HSA+, CD3-, CD4-, CD8-.

Sinclair miniature swine: an animal model of human melanoma.

Sinclair swine display cutaneous melanoma lesions and develop a generalized depigmentation subsequent to tumor regression. Sinclair swine represent a valuable animal model to study the factors influencing the development of melanoma and also the factors which lead to the development of vitiligo. Therefore, information obtained in studies of Sinclair swine should facilitate our understanding of the mechanisms by which melanoma and vitiligo develop and provide us with possible therapeutic treatments for these human diseases.

Summary of first round cluster analysis: complete antibody panel.

The reactivities of 141 monoclonal antibodies (mAbs) with 60 target cell types or lines were analysed by flow cytometry and the data subjected to statistical clustering. mAbs were assigned to 23 clusters by statistical similarity and information on specificity supplied by the contributor. Clustered mAbs were examined in more detail in the second round of workshop analyses.

Report on the behaviour of monoclonal antibodies in the First International Pig CD Workshop identifying the Null cell families.

Clustering analyses were carried out on data from five independent laboratories testing 22 monoclonal antibodies (mAbs) reacting with CD2-sIg-lymphocytes on 14 pig blood and/or tissue lymphoid target cells using cytofluorometry. This was coupled with extensive further studies on blood lymphocytes from normal and thymectomised SLAb/b inbred pigs. These mAbs formed two groups: those mainly identifying the large blood-borne thymus-dependent Null T cells (N) and those reacting with tissue and a small number of blood-borne thymus-independent lymphocytes (N'). Based on their tissue cell reaction patterns, the 10 N' mAbs formed three main groups: N'1A and B; N'2A and B; and N'3. The 12N mAbs fell into four groups N4-N7; N6 was divided into subgroups A-D. One N' (032) and two N mAbs (010 and 063) were unclustered. Based on these data, swine workshop cluster numbers were designated to groups N5 (021, 022 and 059) as SWC4, N6 (061 and 117) as SWC5 and N7 (020 and 141) as SWC6, the latter exceptionally as a single antibody, MAC320, since it is the 'type' mAb identifying effectively all blood Null T lymphocytes. Future research and workshops will have to define with a wider range of techniques the relationships, molecular properties and functional roles of the several new, perhaps novel, antigens identified by this family of fascinating, as yet still poorly defined, mAbs.

Partial cloning and sequencing of the gene encoding the porcine T-cell receptor delta-chain constant region.

The polymerase chain reaction (PCR) was utilized to clone the pig T-cell receptor (TCR) delta-chain constant region-encoding gene (C delta). A cDNA was generated from total RNA preparations of normal pig peripheral blood lymphocytes (PBLs) and a miniature pig peripheral blood cell line (PBLCL 62.G4). The cDNA was used to amplify the porcine TCR C delta gene by PCR using primers chosen by comparing other known C delta sequences for sequence identity. Clones were sequenced and used to determine the primary structure of the porcine TCR C delta chain. A comparison of the nucleotide and deduced amino acid (aa) sequences with the known human, mouse, sheep and cattle sequences revealed that the primary structure of the pig TCR C delta chain has been highly conserved. The immunoglobulin (Ig) domain has two conserved Cys residues and contains a high degree of sequence identity, whereas the hinge region is marked by a high level of diversity. The transmembrane and cytoplasmic regions are also highly conserved, including the presence of the two basic aa, Arg and Lys, in the transmembrane domain. Southern blot analysis has confirmed the presence of one TCR C delta gene in the porcine genome, consistent with similar findings in other species. Thus, the successful cloning and sequencing of the porcine TCR C delta gene should facilitate our understanding of the role of gamma delta T-lymphocytes in the swine immune system.

Isolation and characterization of gamma delta T lymphocyte cell lines from Sinclair swine peripheral blood.

Sinclair miniature swine represent a breed of miniature swine which display a significant incidence of inheritable melanoma which undergo a developmentally regulated spontaneous regression. In an attempt to characterize the host cellular immune response to the melanoma, lymphocyte cell lines have been generated from peripheral blood and designated as peripheral blood lymphocyte cell lines (PBLCLs). The cell lines were expanded in vitro without the addition of exogenous mediators, cloned by limiting dilution, and characterized by flow microfluorimetry, Western, and Northern blot analysis. The cell lines were shown to be CD2-, CD4-, CD8-, and slg-, a phenotype consistent with a null cell population described in swine. The null cell population in swine has been reported to consist of a subpopulation of cells which express the gamma delta T cell receptor (TCR) heterodimer, swine gamma delta T lymphocytes. The PBLCLs were further analyzed by flow microfluorimetry and observed to express the IL-2R, swine MHC Class II antigens, and the endothelial lymphocyte adhesion marker (CD44), which can function as a homing receptor for the skin. In addition, the PBLCLs were observed to express the antigen which is recognized by mAb 86D, an antibody that has been reported to recognize an external epitope on a subset of gamma delta TCR bearing swine T lymphocytes. Western blot analysis of Triton X-114 phase fractions of a PBLCL revealed a protein recognized by the W6 antibody, an antibody which recognizes a conserved region of the C delta chain. Furthermore, Southern and Northern blot analysis indicated that the PBLCL have rearranged the TCR gamma chain gene and express mRNA from the TCR gamma and delta chain genes prior to and following treatment with ionomycin or Concanavalin A. Therefore, the data indicates that the PBLCLs represent swine gamma delta T lymphocyte cell lines which should enable us to enhance our understanding of the role of gamma delta T lymphocytes in the porcine immune system.

Evaluation of the mitogen-induced proliferation and cell surface differentiation antigens of lymphocytes from pigs 1 to 30 days of age.

A study was conducted to characterize aspects of the immune system in the pig from 1 to 30 d of age. Pigs were killed on d 1 (n = 6), d 18 or 19 (n = 6), and d 27 to 30 (n = 7) of age. Lymphocytes were isolated from the blood, thymus, and spleen. Lymphocyte function was assessed for ability to proliferate as induced by concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). Cell surface differentiation antigens on the lymphocytes were evaluated for percentages of cells expressing CD2+, CD4+, CD8+, and SLA class II molecules. Responses of lymphocytes from blood, thymus, and spleen to any of the mitogens were relatively low d 1 through 16; the greatest proliferation occurred by d 28. No detectable percentages of the cell surface differentiation antigens were found on d 1 and changes varied with age and organ. The results indicate that the porcine immune system is not fully developed at birth and the expression of cell surface differentiation antigens seems to occur before the lymphocytes have the ability to respond to mitogens.

Selective activation of murine V beta 8.2 bearing T cells by Pseudomonas exotoxin A.

We have determined that Pseudomonas aeruginosa exotoxin A (PE) can selectively stimulate the proliferation of V beta bearing T lymphocytes. Murine thymocytes were fractionated by selective agglutination with peanut agglutinin (PNA) and the PNA- thymocytes, which represent mature thymocytes, were shown to be responsive to PE stimulation. In addition, mature peripheral T lymphocytes (nylon wool nonadherent splenocytes) were also observed to respond to PE stimulation. Both CD4+ and CD8+ splenic T lymphocyte populations proliferated in response to PE. Flow microfluorimetry analysis of PNA- thymocytes stimulated with PE indicated that V beta 8.2 bearing T cells were preferentially expanded. Thus, our data indicate that PE represents a microbial super antigen which stimulates murine thymocytes which bear the V beta 8.2 element of the T cell receptor.

Dietary fat influences Ia antigen expression and immune cell populations in the murine peritoneum and spleen.

Peritoneal cells (PEC) and splenocytes were obtained from Listeria monocytogene (LM)-infected or noninfected mice fed a 20% fat diet rich in either (n-3) polyunsaturated fatty acids [(n-3) PUFA diet], linoleate [(n-6) PUFA diet], oleate (MONO diet), or saturated fatty acids (SAT diet) for 6 wk and were assessed for T cells, B cells, macrophages and Ia expression by flow cytometric analysis. In the peritoneum of noninfected mice, dietary fat did not affect total cell yield or the percentage of B cells, macrophages or Ia+ cells, but the (n-3) PUFA-fed group had a greater percentage of T cells than did the other groups. Among the LM-infected mice, the (n-3) PUFA-fed group generally had the highest percentage of B cells and the lowest percentages of T cells, macrophages and Ia+ cells in the peritoneum. Listeria monocytogene infection elevated peritoneal T cell numbers in all mice except the (n-3) PUFA-fed group. The density of Ia molecules on PEC was 40% lower in mice fed the (n-3) PUFA diet. In the spleen, dietary fat also influenced the immune cell populations and Ia+ cells. Two-color staining of spleen cells revealed that Ia+ splenocytes were predominately B cells. These data demonstrate that dietary fats influence Ia expression and immune cell populations and that the effects observed in one immune tissue or cell type may not be readily extrapolated to others.

Lymphoproliferative activity of Pseudomonas exotoxin A is dependent on intracellular processing and is associated with the carboxyl-terminal portion.

Pseudomonas aeruginosa exotoxin A (PE) represents a microbial superantigen that requires processing by accessory cells in order to induce the proliferation of V beta 8-bearing murine T lymphocytes. In this study, we have observed that PE requires intracellular processing by a protease in order to induce lymphoproliferation. Pepstatin A, an inhibitor of acid proteases, inhibited PE-induced lymphoproliferation, whereas leupeptin, an inhibitor of serine and thiol proteases, had no effect on PE-induced lymphoproliferation. A number of mutant forms of PE were examined for their ability to induce lymphoproliferation. The mutant form which lacks amino acids 5 to 224 of the receptor-binding domain, PE43, was capable of inducing murine thymocytes to proliferate in the presence of accessory cells. However, neither PEgly276, a mutant toxin which undergoes a different intracellular processing pattern than wild-type PE, nor PE589, a mutant toxin which lacks amino acids 590 to 613 at the carboxyl terminus, was able to induce thymocyte proliferation. In addition, the lymphoproliferation induced by the PE43 mutant form of PE could also be inhibited by pepstatin A. Therefore, our data indicate that intracellular processing by a proteolytic enzyme which is inhibited by pepstatin A is critical for PE-induced lymphoproliferation. Furthermore, the lymphoproliferative activity of PE is associated with the carboxyl-terminal portion of PE.