Vascular lesions in pigs experimentally infected with porcine circovirus type 2 serogroup B.

Vasculitis is a hallmark lesion of the severe form of systemic porcine circovirus-associated disease (PCVAD). In 2 experimental studies with porcine circovirus type 2 serogroup b (PCV2b), 2 pigs developed fatal PCVAD with acute vasculitis, and 5 related pigs developed chronic lymphohistiocytic and plasmacytic peri- and endarteritis. Five of these pigs (1 with acute vasculitis and 4 with chronic vasculitis) had also been inoculated with bovine viral diarrhea virus type 1 (BVDV1) or BVDV1-like virus. Vascular lesions were similar, independent of whether pigs had been inoculated singly with PCV2b or dually with PCV2b and BVDV1 or BVDV1-like virus. The acute vasculitis was accompanied by marked pulmonary and mesenteric edema and pleural effusion. In situ hybridization demonstrated abundant intracytoplasmic porcine circovirus type 2 (PCV2) nucleic acid in endothelial, smooth muscle-like, and inflammatory cells within and around affected arteries. The pigs with lymphohistiocytic and plasmacytic vasculitis had lesions of systemic PCVAD, including multisystemic lymphoplasmacytic and histiocytic or granulomatous inflammation. PCV2 nucleic acid was detected in renal tubule epithelial cells, mononuclear inflammatory cells, and rare endothelial cells in noninflamed vessels in multiple tissues of these animals. The 2 pigs with acute vasculitis had no PCV2-specific antibodies (or a low titer of), whereas the pigs with lymphohistiocytic and plasmacytic vasculitis developed high antibody titers against this virus. These observations suggest that (1) acute vasculitis observed in the current studies is directly caused by PCV2b, (2) chronic vasculitis may in part be mediated by the subsequent immune response, and (3) host factors and viral strain may both contribute to vasculitis in animals infected with PCV2b.

Sebaceous adenocarcinoma of the external auditory canal in a New Zealand white rabbit.

A rare sebaceous gland carcinoma of the external auditory canal in a rabbit is described. The tumour was characterized histologically by foci and cords of markedly pleomorphic cells with abundant cytoplasm and variable numbers of vacuoles. A single pulmonary mass had similar histological characteristics. This is the first such tumour reported in a rabbit.

Encapsulation of recombinant adenovirus into alginate microspheres circumvents vector-specific immune response.

Pre-existing immunity against adenoviruses may compromise the efficacy of adenoviral vectors for vaccination and gene therapy. The purpose of this study was to determine whether encapsulation of adenovirus recombinants into biodegradable alginate microparticles could circumvent the vector-specific immune response. Mice were immunized either intranasally (i.n.) or intraperitoneally (i.p.) with human adenovirus type 5 (HAd5), resulting in the development of virus-specific antibodies. Immunized and nai;ve mice were inoculated with AdCA36lacZ (an E1-deleted HAd5 recombinant containing the bacterial beta-galactosidase (LacZ) gene), encapsulated (E) into alginate microparticles, or nonencapsulated (NE) ie, as a virus suspension. LacZ expression in animals immunized once (1x) or twice (2x) with HAd5 and subsequently inoculated with NE-AdCA36lacZ (NE-Z) was significantly (P<0.001) reduced compared to those levels observed in NE-Z inoculated nai;ve mice, suggesting that the immune response against the vector adversely affected transgene expression. In contrast, there was only slight reduction (P>0.05) in LacZ expression in mice immunized 1x or 2x with HAd5 that were subsequently inoculated with E-AdCA36lacZ (E-Z) compared to those levels obtained in E-Z inoculated nai;ve animals. Similar results were obtained with i.n. or i.p. inoculated animals. These results indicate that microencapsulation of recombinant adenovirus effectively circumvented the vector-specific immune response.

Induction of systemic and mucosal immune response in cattle by intranasal administration of pig serum albumin in alginate microparticles.

Biodegradable microparticles are an efficient mucosal delivery system that protect antigens from the harsh mucosal environment and facilitate their uptake by M cells at the epithelium of mucosal-associated lymphoid tissue. In this study, we determined the systemic and mucosal immune response in calves following intranasal and oral immunization with pig serum albumin (PSA) encapsulated in alginate microparticles. The size of the particles ranged from 1 to 50 microm in diameter, with 95% of the particles being smaller than 5 microm. High levels of anti-PSA IgG1 antibodies were found in the serum, nasal secretions, and to a less extent in saliva of calves vaccinated intranasally, but not orally, with PSA-microparticles. There was no significant increase of PSA-specific IgA. A weak lymphocyte proliferative immune response was observed in peripheral blood mononuclear cells (PBMCs), and few anti-PSA antibody-secreting cells (ASC) were detected in the blood of calves immunized intranasally. The combined systemic and mucosal response observed in intranasally immunized animals may be attributed to the wide variation in the size of the alginate microparticles, with smaller particles translocating to regional lymph nodes and inducing a systemic immune response, and larger particles being retained in the NALT and inducing a mucosal immune response. The procedure presented here may be useful as an intranasal vaccine against respiratory diseases in cattle.

Kinetics and type of immune response following intranasal and subcutaneous immunisation of calves.

Protection of animals against respiratory infections has long been known to depend on respiratory mucosal immunity. However, few studies have been reported on the immune response following intranasal (i.n.) immunisation with non-living, soluble antigens. This study determined the kinetics of the humoral and cellular immune responses in calves after i.n. immunisation with Limulus haemocyanin (LH) with cholera toxin adjuvant, or subcutaneous (s.c.) immunisation with LH in incomplete Freund's adjuvant. A proliferative response of peripheral blood mononuclear cells cultured in vitro with LH was observed in animals immunised 7-10 days after i.n. and s.c. immunisations with no significant differences between the two immunised groups. LH -specific antibody was present in the serum of animals immunised s.c. (IgM, IgG1 and IgG2) and i.n. (IgA). Although significant IgA responses were observed, i.n. immunisations in cattle with soluble protein antigens and cholera toxin as an adjuvant did not induce a strong systemic immune response.

Change in the degree of adsorption of proteins by aluminum-containing adjuvants following exposure to interstitial fluid: freshly prepared and aged model vaccines.

The ability of interstitial fluid to change the degree of adsorption of ovalbumin to aluminum hydroxide adjuvant or lysozyme to aluminum phosphate adjuvant was studied. Ovalbumin and lysozyme were almost completely eluted after exposure at 37 degrees C to sheep lymph fluid for 4h or 15 min, respectively. The ability of sheep lymph fluid to elute lysozyme from aluminum phosphate adjuvant did not change as the model vaccine aged. However, only 60% of the ovalbumin adsorbed to aluminum hydroxide adjuvant was eluted during exposure to sheep lymph fluid for 24h after the model vaccine aged for 11 weeks at 4 degrees C.

Detoxification of endotoxin by aluminum hydroxide adjuvant.

Langmuir adsorption isotherms of endotoxin and aluminum-containing adjuvants at pH 7.4 and 25 degrees C revealed that aluminum hydroxide adjuvant has a greater adsorption capacity (283 microg/mg Al) and adsorption coefficient (1.3x10(4) ml/miccrog) than aluminum phosphate adjuvant (3.0 microg/mg Al, 0.20 ml/microg). The difference in endotoxin adsorption was related to two adsorption mechanisms: electrostatic attraction and covalent bonding. The isoelectric point (iep) of endotoxin is approximately 2. An electrostatic attractive force will be present with aluminum hydroxide adjuvant (iep=11.4), and an electrostatic repulsive force will operate with aluminum phosphate adjuvant (iep=4.6). Endotoxin contains two phosphate groups in the lipid A portion. Covalent bonding occurs with surface aluminum in aluminum hydroxide adjuvant but is inhibited by surface phosphate in aluminum phosphate adjuvant. In-vitro desorption experiments using components of interstitial fluid showed that endotoxin adsorbed by aluminum hydroxide adjuvant was not desorbed by interstitial anions (5 mM phosphate or 2.7 mM citrate) or interstitial proteins (25 mg albumin/ml). The effect of aluminum-containing adjuvants on the systemic response of Sprague-Dawley rats to a 15 microg/kg subcutaneous dose of endotoxin was determined by measuring the serum concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). TNF-alpha and IL-6 were observed in the group which received an endotoxin solution or endotoxin and aluminum phosphate adjuvant. No TNF-alpha or IL-6 was detected in the group that received endotoxin and aluminum hydroxide adjuvant. Aluminum hydroxide adjuvant detoxifies endotoxin by adsorbing it in the vaccine and then not releasing it in interstitial fluid upon administration.

Modulation of allergic inflammation in mice deficient in TNF receptors.

Tumor necrosis factor-alpha (TNF) is implicated as an important proinflammatory cytokine in asthma. We evaluated mice deficient in TNF receptor 1 (TNFR1) and TNFR2 [TNFR(-/-) mice] in a murine model of allergic inflammation and found that TNFR(-/-) mice had comparable or accentuated responses compared with wild-type [TNFR(+/+)] mice. The responses were consistent among multiple end points. Airway responsiveness after methacholine challenge and bronchoalveolar lavage (BAL) fluid leukocyte and eosinophil numbers in TNFR(-/-) mice were equivalent or greater than those observed in TNFR(+/+) mice. Likewise, serum and BAL fluid IgE; lung interleukin (IL)-2, IL-4, and IL-5 levels; and lung histological lesion scores were comparable or greater in TNFR(-/-) mice compared with those in TNFR(+/+) mice. TNFR(+/+) mice chronically treated with anti-murine TNF antibody had BAL fluid leukocyte numbers and lung lesion scores comparable to control antibody-treated mice. These results suggest that, by itself, TNF does not have a critical proinflammatory role in the development of allergic inflammation in this mouse model and that the production of other cytokines associated with allergic disease may compensate for the loss of TNF bioactivity in the TNFR(-/-) mouse.

Immunization with DNA, adenovirus or both in biodegradable alginate microspheres: effect of route of inoculation on immune response.

To determine the potential for biodegradable alginate microspheres to be used as a delivery vehicle for DNA based vaccines, we constructed a plasmid, pMNe-gal-SV40, containing the bacterial beta-galactosidase (LacZ) gene under the control of the murine cytomegalovirus (MCMV) immediate-early promoter and the simian virus 40 (SV40) polyadenylation signal. The effect of the route of administration and co-administration of adenovirus on systemic and mucosal immune responses were investigated. Mice were inoculated orally, intranasally (i.n.), intramuscularly (i.m.), subcutaneously (s.c.) or intraperitoneally (i.p.) on days 0, 14 and 28 with microspheres containing plasmid DNA, bovine adenovirus type 3 (BAd3) or plasmid DNA + BAd3. Systemic routes of immunization (i.m., s.c. and i.p.) resulted in higher LacZ- or BAd3-specific IgG ELISA titers compared to those obtained by mucosal routes of inoculation (oral and i.n.). Mucosal immunization led to slightly higher titers of LacZ- or BAd3-specific IgA at mucosal sites compared to those obtained by the various systemic routes. All the routes of immunization induced LacZ-specific lymphoproliferation. Co-administration of BAd3 enhanced the LacZ-specific IgG response irrespective of the route of administration.

Mice lacking the transcription factor RelB develop T cell-dependent skin lesions similar to human atopic dermatitis.

Mice with a targeted disruption of the Rel / NF-kappaB family member RelB develop a complex inflammatory phenotype and hematopoietic abnormalities. RelB-deficient (relB(- / -)) mice were clinically normal until 4 - 10 weeks after birth when thickening of the skin and hair loss developed. Histological and immunohistochemical evaluation of relB(- / -) skin lesions revealed hyperkeratosis and marked epidermal hyperplasia. Many CD4(+) T cells and eosinophils mixed with lesser numbers of CD8(+) T cells and neutrophils were present in the dermis. There was a moderate increase of MHC class II-positive dermal dendritic cells and dermal mast cells. Increased expression of Th2 cytokines correlated with increased mRNA levels of eotaxin and CCR3 in relB(- / -) skin. The dermatitis did not develop in the offspring of relB(- / -) mice crossed with transgenic mice that lack peripheral T cells, demonstrating that the skin lesions were T cell dependent. The dermatitis observed in RelB-deficient mice had many similarities with atopic dermatitis in human patients including infiltrating CD4(+) T cells and eosinophils in the skin, increased number of eosinophils in the blood and increased serum IgE. Thus, the relB(- / -) mouse should be a useful model to study the pathogenesis of this common allergic human disease.

Circumvention of vector-specific neutralizing antibody response by alternating use of human and non-human adenoviruses: implications in gene therapy.

To determine whether non-human adenovirus-specific antibodies are cross-neutralizing, rabbit and mouse anti-human adenovirus type 5 (HAd5), anti-bovine adenovirus type 3 (BAd3), and anti-porcine adenovirus type 3 (PAd3) sera were used in cross-virus neutralization assays. Adenovirus neutralizing antibodies were found to be virus-specific, suggesting that virus neutralizing epitope differs significantly in HAd5, BAd3, and PAd3. To further investigate whether immunity to an HAd5-derived vector could be circumvented by the use of non-human adenoviruses in vivo, mice were first immunized either intranasally or intraperitoneally with HAd5, BAd3, PAd3, or BAd3 + PAd3, and after development of adenovirus-specific antibodies, animals were inoculated with the HAd5 recombinant (AdCA36lacZ) containing the bacterial beta-galactosidase gene under the control of murine cytomegalovirus immediate-early promoter. Virus-inoculated animals developed virus-specific IgG and IgA antibodies. LacZ expression in animals initially primed with HAd5 was significantly reduced (P < 0.05), suggesting that the immune response against the vector could prevent the transgene expression following subsequent inoculation of the same vector, whereas LacZ expression in mice initially primed with BAd3, PAd3, or BAd3 + PAd3 was significantly higher (P > 0.05) than that obtained in HAd5-primed animals. Our results suggest that HAd5-, BAd3-, or PAd3-based vectors may be used sequentially for human gene therapy or vaccine production as a means to avoid immunity to the vector.

Biodegradable alginate microspheres as a delivery system for naked DNA.

Sodium alginate is a naturally occurring polysaccharide that can easily be polymerized into a solid matrix to form microspheres. These biodegradable microspheres were used to encapsulate plasmid DNA containing the bacterial beta-galactosidase (LacZ) gene under the control of either the cytomegalovirus (CMV) immediate-early promoter or the Rous sarcoma virus (RSV) early promoter. Mice inoculated orally with microspheres containing plasmid DNA expressed LacZ in the intestine, spleen and liver. Inoculation of mice with microspheres containing both the plasmid DNA and bovine adenovirus type 3 (BAd3) resulted in a significant increase in LacZ expression compared to those inoculated with microspheres containing only the plasmid DNA. Our results suggest that adenoviruses are capable of augumenting transgene expression by plasmid DNA both in vitro and in vivo.

Absence of Peyer's patches and abnormal lymphoid architecture in chronic proliferative dermatitis (cpdm/cpdm) mice.

The chronic proliferative dermatitis (cpdm) mutation causes inflammation in multiple organs, most prominently in the skin. Examination of the immune system revealed severe abnormalities in the architecture of lymphoid tissues. Peyer's patches were absent. In contrast, the spleen, lymph nodes, and nasal-associated lymphoid tissues were present. The spleen had normal numbers of T and B cells, but the spleen, lymph nodes, and nasal-associated lymphoid tissues had poorly defined follicles and lacked germinal centers and follicular dendritic cells. The marginal zone in the spleen was absent. The total concentration of serum IgG, IgA, and IgE in cpdm/cpdm mice was significantly decreased, whereas serum IgM was normal. Fecal IgA was low to undetectable in mutant mice, and the concentration of fecal IgM was increased. The titer of DNP-specific Abs following immunization with DNP-keyhole limpet hemocyanin was significantly decreased for all IgG subclasses. In contrast, T cell function appeared normal as assessed by evaluation of the contact hypersensitivity response in cpdm/cpdm mice. The cpdm mutation causes a complex phenotype that is characterized by multiorgan inflammation and the defective development of lymphoid tissues. The cpdm/cpdm mouse may be a useful model to study the factors that control the development of lymphoid tissues, in particular the Peyer's patches, and the mechanisms that control the humoral immune response.

Primary vascular neoplasms of lymph nodes in the dog.

Primary vascular neoplasms of lymph nodes are rare, and appear not to have previously been reported in domestic animals. This report describes hemangiomas and a lymphangioma in lymph nodes of aged Beagle dogs. Eight hemangiomas (4.8%) and one lymphangioma were present in 165 examined popliteal lymph nodes, and one hemangioma occurred in a hepatic lymph node. The hemangiomas were cavernous and benign.

Tenfold increased incidence of spontaneous multiple myeloma in long-term immunosuppressed aging C57BL/KaLwRij mice.

Persons undergoing maintenance immunosuppressive treatment (MIST) were shown to be at increased risk for the development of early malignancies, often of cells of the immune system. Very little is known about the late effects of MIST. Some clinical studies indicated an age-related increase in the incidence of plasma-cell disorders, in particular in that of multiple myeloma (MM). In the present study the influence of MIST on the development of monoclonal B-cell proliferative disorders, monoclonal gammopathies (MG), was studied in an animal model, the C57BL/KaLwRij mouse. This strain is known for its susceptibility to develop with aging MG similar to those in humans. Two widely used treatment protocols (azathioprine/prednisolone and Cyclosporin A/prednisolone) were tested in young and adult mice. Both regiments were shown to increase 10-fold the incidence of spontaneous multiple myeloma. Unexpectedly, the same high incidence of MM and in addition the development of a life-shortening lymphoblastic lymphoma were found in a high frequency in the control group that received Cremophor EL only, i.e., the solvent of Cyclosporin A. Repeated experiments with another lot of Cremophor showed a 6-fold increased frequency of NM but no lymphoblastic lymphoma. With respect to the life-span and the incidence of hemopoietic neoplasms the least harmful drugs for MIST appeared to be azathioprine/prednisolone. The results of the experiments in this C57BL/KaLwRij mouse model give a warning for increased incidence of MM in susceptible aging individuals and address a question whether Cremophor EL is a safe solvent for Cyclosporin A.

Pathogenesis of skin lesions in mice with chronic proliferative dermatitis (cpdm/cpdm).

Chronic proliferative dermatitis is a spontaneous mutation in C57BL/Ka mice (cpdm/cpdm), showing alopecia, epithelial hyperproliferation, infiltration by eosinophils and macrophages, and vascular dilatation. To further elucidate its pathogenesis, organs of 1-, 2-, 3-, 4-, 5-, and 6-week-old cpdm/cpdm mice were examined. At 4 weeks, the epidermal thickness was increased, whereas already at 3 weeks, the bromodeoxyuridine incorporation was increased in the basal keratinocytes. However, already at the age of 1 week, skin, lungs, and lymph nodes were infiltrated by eosinophils although no macroscopic lesions were present. Compared with control animals, 6-week-old cpdm/cpdm mice had decreased serum IgE levels and increased numbers of mast cells. From the age of 1 week these mast cells became increasingly IgE positive. In contrast, the mast cells of the control animals remained IgE negative. Mast cells of control and cpdm/cpdm mice were interleukin-4 and tumor necrosis factor-alpha positive. A likely explanation for the tissue infiltration of eosinophils could be the release of interleukin-4 and tumor necrosis factor-alpha from activated mast cells. Tumor necrosis factor-alpha may lead to the expression of E-selectin on endothelial cells, facilitating interleukin-4-mediated eosinophil transendothelial migration. Although various pathogenetic aspects of the cpdm/cpdm mouse need further elucidation, this model can be a tool to study eosinophil infiltration, leukocyte-endothelial cell interactions, and mast cell proliferation. Furthermore, the cpdm/cpdm mouse can be used to study chronic inflammatory skin disease because of the severe epidermal proliferation.

Interleukin-6 activity in dogs with juvenile polyarteritis syndrome: effect of corticosteroids.

Juvenile polyarteritis syndrome (JPS) is an idiopathic febrile disease in dogs. Elevated serum levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) have been reported in human patients with vasculitis. We investigated whether these cytokines are also elevated in serum of dogs with JPS using sensitive bioassays. Increased levels of IL-6 activity were detected in the serum of 12 acutely ill dogs, whereas the IL-6 activity decreased to low or undetectable levels during convalescence. Treatment of 5 acute JPS dogs with prednisone resulted in a rapid clinical improvement accompanied by a decrease of IL-6 activity. Withdrawal of prednisone treatment caused reappearance of clinical symptoms and high serum IL-6 activity within a few days. TNF activity could not be detected in the samples of normal dogs, convalescent JPS, or acute JPS dogs. These studies support a role for IL-6 in the pathogenesis of JPS.

Pathologic features of naturally occurring juvenile polyarteritis in beagle dogs.

Eighteen young Beagle dogs (eight males and 10 females), ages 6-40 months, with canine juvenile polyarteritis syndrome (CJPS), a naturally occurring vasculitis and perivasculitis of unknown etiology, were necropsied, and their tissues were examined by histopathologic and histochemical methods. The condition is characterized by recurring episodes of an acute onset of fever (> 40 C) and neck pain that persist for 3-7 days. The major histopathologic alterations were a systemic vasculitis and perivasculitis. During the febrile, painful period of CJPS, the vascular lesions ranged from a histiocytic-lymphocytic periarterial infiltration to transmural arterial inflammation with concomitant fibrinoid necrosis and vascular thrombosis. Massive periarterial accumulations of inflammatory cells were common and often extended into adjacent tissues. The small- to medium-sized muscular arteries of the heart, cranial mediastinum, and cervical spinal meninges were consistently involved. Vasculitis occasionally occurred in other organ systems. The vascular lesions in dogs examined during clinically normal periods consisted of intimal and medial fibrosis, ruptured elastic laminae, and mild perivasculitis; these lesions were probably related to previous episodes of vasculitis. Eight dogs that had experienced repeated acute episodes also developed splenic, hepatic, and renal amyloidosis. The clinical signs, laboratory abnormalities, and the vascular lesions suggest that the condition may be immune-system mediated. CJPS may serve as a naturally occurring animal model of human immune-system-mediated vasculitides such as polyarteritis nodosa, infantile polyarteritis, and Kawasaki disease.

Constitutive expression of Ly-6.A2 on murine keratinocytes and inducible expression on TCR gamma delta+ dendritic epidermal T cells.

We investigated the expression of Ly-6.A2 on isolated murine epidermal cells by flow cytometry. Ly-6.A2 was expressed on 61% of keratinocytes and 6% of dendritic epidermal T cells of C57BL mice. Phosphatidylinositol-specific phospholipase C removed Ly-6.A2, indicating that the antigen is anchored to the keratinocyte membrane via a glycosyl-phosphatidylinositol anchor similar to its attachment to the membrane of lymphocytes. Induction of dermatitis by topical application of PMA increased the expression of Ly-6.A2 on TCR gamma delta+ dendritic epidermal T cells and did not change its expression on keratinocytes. The increased expression of Ly-6.A2 on dendritic epidermal T cells was transient and reached a peak at 4 days after application of PMA, when 55% of the cells were positive.

Gastrointestinal AAPOAII and systemic AA-amyloidosis in aged C57BL/Ka mice. Amyloid-type dependent effect of long-term immunosuppressive treatment.

The light microscopic and immunohistochemical features of a novel localized senile amyloidosis in the gastrointestinal tract of C57BL/Ka mice are described. Senile gastrointestinal amyloidosis was predominantly found in the lamina propria of the ileum, cecum and stomach and infrequently in other segments of the gastrointestinal tract. The Congo red affinity of the senile amyloid was sensitive to potassium permanganate pretreatment. The amyloid did not react with anti-AA and anti-immunoglobulin antisera, but stained positively for apoAII, a major apolipoprotein of high density lipoproteins. A similar type of amyloid, termed AApoAII, has recently been described in a systemic form of senile amyloidosis in mice. In the present study, we investigated the effect of long-term immunosuppressive treatment on the incidence of systemic AA-amyloidosis and gastrointestinal AApoAII-amyloidosis in aged C57BL/Ka mice. Gastrointestinal amyloidosis occurred in 60% of the control mice, but significantly less in mice of the immunosuppressed groups. In contrast, systemic AA-immunoreactive amyloidosis was only found in mice that were given immunosuppressive treatment. There was no codeposition of AA and AApoAII-amyloid. These findings indicate that immunosuppressive drugs have a profound effect on the incidence as well as the type of amyloidosis in C57BL/Ka mice.