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Intradermal (ID) immunization is an attractive route of vaccination because it targets tissue rich in dendritic cells, has dose-sparing potential, and allows needle-free delivery. However, few adjuvants are effective, nonreactogenic, and compatible with needle-free delivery devices. In this study, we demonstrate that a combination adjuvant composed of cyclic-di-AMP (cdAMP) and the plant-derived nanoparticle adjuvant Nano-11 significantly enhanced the immune response to ID-injected vaccines in mice and pigs with minimal local reaction at the injection site. The cdAMP/Nano-11 combination adjuvant increased Ag uptake by lymph node–resident and migratory skin dendritic cell subpopulations, including Langerhans cells. ID immunization with cdAMP/Nano-11 expanded the population of germinal center B cells and follicular helper T cells in the draining lymph node and Ag-specific Th1 and Th17 cells in the spleen. It elicited an enhanced immune response with a significant increase of IgG1 and IgG2a responses in mice at a reduced dose compared with i.m. immunization. An increased IgG response was observed following needle-free ID immunization of pigs. Nano-11 and cdAMP demonstrated a strong synergistic interaction, as shown in the activation of mouse, human, and porcine APC, with increased expression of costimulatory molecules and secretion of TNF and IL-1β. The combination adjuvant induced robust activation of both NF-κB and IFN regulatory factor signaling pathways and the NLRP3 inflammasome. We conclude that the combination of Nano-11 and cdAMP is a promising adjuvant for ID delivery of vaccines that supports a balanced immune response.

Subunit vaccines generally require adjuvants to achieve optimal immune responses. Toll-like receptor (TLR) agonists are promising immune potentiators, but rapid diffusion from the injection site reduces their local effective concentration and may cause systemic reactions. In this study, we investigated the potential of aluminum hydroxide adjuvant (AH) to adsorb the TLR3 agonist poly(I:C) and TLR9 agonist CpG and compared the effect of the combination adjuvant on the immune response with either the TLR agonists or AH alone in mice. Poly(I:C) and CpG readily adsorbed onto AH and this combination adjuvant induced a stronger IgG1 and IgG2a immune response with a significant increase of antibody avidity. The combination adjuvant enhanced antigen uptake and activation of dendritic cells in vitro. It induced an inflammatory response at the injection site similar to AH but without eosinophils which are typically observed with AH. A distinctive antigen-containing monocyte/macrophage population with an intermediate level of CD11c expression was identified in the draining lymph nodes after immunization with TLR agonists and the combination adjuvant. Injection of the combination adjuvant did not induce an increase of TNFα and CXCL10 in serum in contrast to the injection of soluble TLR agonists. These results indicate that this combination adjuvant is a promising formulation to solve some of the unmet needs of current vaccines.

Spontaneous mutations in the SHANK-associated RH domain interacting protein (Sharpin) resulted in a severe autoinflammatory type of chronic proliferative dermatitis, inflammation in other organs, and lymphoid organ defects. To determine whether cell-type restricted loss of Sharpin causes similar lesions, a conditional null mutant was created. Ubiquitously expressing cre-recombinase recapitulated the phenotype seen in spontaneous mutant mice. Limiting expression to keratinocytes (using a Krt14-cre) induced a chronic eosinophilic dermatitis, but no inflammation in other organs or lymphoid organ defects. The dermatitis was associated with a markedly increased concentration of serum IgE and IL18. Crosses with S100a4-cre resulted in milder skin lesions and moderate to severe arthritis. This conditional null mutant will enable more detailed studies on the role of SHARPIN in regulating NFkB and inflammation, while the Krt14-Sharpin-/- provides a new model to study atopic dermatitis.

Intranasal vaccination elicits secretory IgA (SIgA) antibodies in the airways, which is required for cross-protection against influenza. To enhance the breadth of immunity induced by a killed swine influenza virus antigen (KAg) or conserved T cell and B cell peptides, we adsorbed the antigens together with the TLR3 agonist poly(I:C) electrostatically onto cationic alpha-D-glucan nanoparticles (Nano-11) resulting in Nano-11-KAg-poly(I:C) and Nano-11-peptides-poly(I:C) vaccines. In vitro, increased TNF-&alpha, and IL-1&szlig, cytokine mRNA expression was observed in Nano-11-KAg-poly(I:C)-treated porcine monocyte-derived dendritic cells. Nano-11-KAg-poly(I:C), but not Nano-11-peptides-poly(I:C), delivered intranasally in pigs induced high levels of cross-reactive virus-specific SIgA antibodies secretion in the nasal passage and lungs compared to a multivalent commercial influenza virus vaccine administered intramuscularly. The commercial and Nano-11-KAg-poly(I:C) vaccinations increased the frequency of IFN&gamma, secreting T cells. The poly(I:C) adjuvanted Nano-11-based vaccines increased various cytokine mRNA expressions in lymph nodes compared to the commercial vaccine. In addition, Nano-11-KAg-poly(I:C) vaccine elicited high levels of virus neutralizing antibodies in bronchoalveolar lavage fluid. Microscopic lung lesions and challenge virus load were partially reduced in poly(I:C) adjuvanted Nano-11 and commercial influenza vaccinates. In conclusion, compared to our earlier study with Nano-11-KAg vaccine, addition of poly(I:C) to the formulation improved cross-protective antibody and cytokine response.

Cell-mediated immune responses characterized by the secretion of IFNγ and IL-17 play an important role in the immune response to Bordetella pertussis (B. pertussis). We investigated innate sources of IFNγ and IL-17 upon stimulation of spleen cells from BALB/c (B/c) and C57BL/6 (B6) mice with heat-killed B. pertussis (hkBp). Spleen cells from B/c mice secreted less IFNγ and more IL-17 than those from B6 mice. Innate IFNγ was produced predominantly by NK cells in B/c mice and by CD8 T cells and NK cells in B6 mice. Innate IL-17 was produced primarily by γδT cells in both mouse strains. The secretion of IFNγ was abrogated by anti-IL-12, and the production of IL-17 was abolished by anti-IL-1β- and anti-IL23-neutralizing antibodies. B/c dendritic cells (DCs) stimulated with hkBp secreted significantly more IL-1β and less IL-12 than B6 DCs. Differences in JNK phosphorylation in DCs suggest that this pathway plays a role in the differences between B/c and B6 strains. Mixed cultures of DCs and γδT cells from B/c and B6 showed that cytokines from DCs as well as γδT cell-intrinsic factors contributed to the robust innate IL-17 response in B/c strain. Stimulation of γδT cells with IL-1β and IL-23 was sufficient for IL-17 secretion whereas IL-12 inhibited the secretion of IL-17. A larger fraction of γδT cells were γδT-17 cells in B/c mice than B6 mice. Our data indicate important roles for genetically determined factors in the innate IFNγ and IL-17 responses to B. pertussis.

Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and profoundly affect host health and disease. SCFAs generate IL-10+ regulatory T cells, which may promote immune tolerance. However, SCFAs can also induce Th1 and Th17 cells upon immunological challenges and, therefore, also have the potential to induce inflammatory responses. Because of the seemingly paradoxical SCFA activities in regulating T cells, we investigated, in depth, the impact of elevated SCFA levels on T cells and tissue inflammation in mice. Orally administered SCFAs induced effector (Th1 and Th17) and regulatory T cells in ureter and kidney tissues, and they induced T cell–mediated ureteritis, leading to kidney hydronephrosis (hereafter called acetate-induced renal disease, or C2RD). Kidney hydronephrosis in C2RD was caused by ureteral obstruction, which was, in turn, induced by SCFA-induced inflammation in the ureteropelvic junction and proximal ureter. Oral administration of all major SCFAs, such as acetate, propionate, and butyrate, induced the disease. We found that C2RD development is dependent on mammalian target of rapamycin activation, T cell–derived inflammatory cytokines such as IFN-γ and IL-17, and gut microbiota. Young or male animals were more susceptible than old or female animals, respectively. However, SCFA receptor (GPR41 or GPR43) deficiency did not affect C2RD development. Thus, SCFAs, when systemically administered at levels higher than physiological levels, cause dysregulated T cell responses and tissue inflammation in the renal system. The results provide insights into the immunological and pathological effects of chronically elevated SCFAs.

Biodegradable nanoparticles with functionalized surfaces are attractive candidates as vaccine adjuvants. Nano-11 are cationic dendrimer-like α-D-glucan nanoparticles with a diameter of 70–80 nm. Mice injected with antigen formulated with Nano-11 developed antibody titers that were similar or greater than antigen with aluminum adjuvant. Utilizing an in vivo imaging system, Nano-11 was shown to remain at the injection site after administration and cleared gradually over the course of 3 weeks. Injection of Nano-11 induced a transient inflammatory response characterized by recruitment of a mixed population of inflammatory cells, predominantly monocytes and macrophages with relatively few neutrophils. Recruited Mac-2+macrophages efficiently phagocytized the majority of Nano-11 at the injection site. Fluorescently labeled Nano-11 was present in cells in the draining lymph nodes 1 day after injection, with the majority contained in migratory dendritic cells. Injection of ovalbumin adsorbed to Nano-11 resulted in an increase of ovalbumin-containing cells in draining lymph nodes. Nano-11 delivered more antigen to antigen-presenting cells on a per cell basis and demonstrated more specific targeting to highly immunopotentiating migratory dendritic cells compared with soluble or aluminum hydroxide adsorbed ovalbumin. These results support the efficacy of Nano-11 and its potential use as a next generation vaccine adjuvant., Adjuvants: Discovering the next generation of vaccine boosters A plant-derived nanoparticle boosts the immune system’s response to foreign stimuli, such as vaccines, through newly discovered mechanisms. Harm HogenEsch and his team from Purdue University, USA, previously discovered that their formulated nanoparticle adjuvant, Nano-11, increased immune responses to stimuli in mice. Adjuvants are formulations often co-administered with vaccines to promote interaction with the host’s immune system, conferring greater protection. In this study, the scientists found that Nano-11 functions by inducing a temporary inflammation, attracting immune cells to the injection site. The nanoparticles also facilitate the transport of the stimuli to the lymph nodes. Nano-11 is metabolized without deposition in any major internal organs, suggesting a positive safety profile. As current aluminum-based adjuvants are sometimes poorly effective and can cause local adverse reactions, nanoparticle-based potentiators such as Nano-11 may form the next generation of adjuvants.

Mouse models of various types of inflammatory skin disease are often accompanied by increased dermal angiogenesis. The C3H/HeJ inbred strain spontaneously develops alopecia areata (AA), a cell mediated autoimmune disorder that can be controllably expanded using full thickness skin grafts to young unaffected mice. This provides a reproducible and progressive model for AA in which the vascularization of the skin can be examined. Mice receiving skin grafts from AA or normal mice were evaluated at 5, 10, 15, and 20 weeks after engraftment. Lymphatics are often overlooked as they are small slit-like structures above the hair follicle that resemble artifact-like separation of collagen bundles with some fixatives. Lymphatics are easily detected using lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) by immunohistochemistry to label their endothelial cells. Using LYVE1, there were no changes in distribution or numbers of lymphatics although they were more prominent (dilated) in the mice with AA. Lyve1 transcripts were not significantly upregulated except at 10 weeks after skin grafting when clinical signs of AA first become apparent. Other genes involved with vascular growth and dilation or movement of immune cells were dysregulated, mostly upregulated. These findings emphasize aspects of AA not commonly considered and provide potential targets for therapeutic intervention.

Myelodysplastic syndrome is a clonal process characterized by ineffective hematopoiesis and progression to acute leukemia. Although many myelodysplastic syndrome and leukemic patients have compromised immunity, the role of underlying mutations in regulating immune function is poorly understood. Recent studies show that NUP98-HOXD13 (NHD13) fusion gene results in myelodysplastic syndrome and impairs lymphocyte differentiation in transgenic mice. In our studies, we sought to elucidate the mechanism by which NHD13 affects B-lymphocyte development and function. Based on our preliminary findings that transgenic mice had increased levels of IgM and reduced IgG1 and IgE, we hypothesized that the fusion gene might impair class switch recombination (CSR). Mice were immunologically challenged with dinitrophenol. NHD13 mice showed a marked reduction in B-lymphocyte differentiation in their bone marrow and spleen following dinitrophenol stimulation and had reduced production of dinitrophenol-specific antibodies. Spleen follicles from these mice were small and hypocellular, indicating failure of clonal expansion. When isolated NHD13 B lymphocytes were stimulated in vitro using Escherichia coli lipopolysaccharide or lipopolysaccharide + interleukin-4, they failed to undergo sufficient CSR and proliferation. Taken together, our findings show that expression of NUP98-HOXD13 impairs CSR and reduces the antibody-mediated immune response, in addition to its role in leukemia. Further delineation of the NUP98-HOXD13 transgene may reveal novel pathways involved in CSR.

Th17 cells are major effector T cells in the intestine, but the regulation of their tissue tropism within the gut is poorly understood. We investigated the roles of vitamin A and retinoic acid in generation of inflammatory Th17 cells with distinct tissue tropisms within the intestine. We found that Th17 cells with distinct tissue tropisms and pathogenic activities are generated depending on the available concentration of retinoic acid (RA). In contrast to the widespread perception that RA would suppress the generation of Th17 cells, we provide evidence that RA is actually required for generation of Th17 cells with specific tissue tropisms within the gut. Th17 cells induced at suboptimal serum concentrations of RA migrated and induced moderate inflammation mainly in the large intestine, whereas the Th17 cells induced with optimal levels of exogenous RA (∼10 nM) migrated to the small intestine and induced more severe inflammation. The Th17 cells, induced in the presence or absence of RA, differentially expressed the trafficking receptors CCR9 and α4β7. CCR9 is required for Th17 cell migration to the small intestine, whereas α4β7 is required for the migration of Th17 cells throughout the whole intestine. Our results identified RA as a major signal that regulates the generation of gut Th17 cells with distinct capacities in migration and inflammatory activities. The results indicate also that specific gut tropism of Th17 cells is determined by the combination of trafficking receptors regulated by the RA signal.

Severe sepsis is one of the leading causes of death worldwide. High mortality rates in sepsis are frequently associated with neutropenia. Despite the central role of neutrophils in innate immunity, the mechanisms causing neutropenia during sepsis remain elusive. Here, we show that neutropenia is caused in part by apoptosis and is sustained by a block of hematopoietic stem cell (HSC) differentiation. Using a sepsis murine model, we found that the human opportunistic bacterial pathogen Pseudomonas aeruginosa caused neutrophil depletion and expansion of the HSC pool in the bone marrow. “Septic” HSCs were significantly impaired in competitive repopulation assays and defective in generating common myeloid progenitors and granulocyte-monocyte progenitors, resulting in lower rates of myeloid differentiation in vitro and in vivo. Delayed myeloid-neutrophil differentiation was further mapped using a lysozyme–green fluorescent protein (GFP) reporter mouse. Pseudomonas's lipopolysaccharide was necessary and sufficient to induce myelosuppresion and required intact TLR4 signaling. Our results establish a previously unrecognized link between HSC regulation and host response in severe sepsis and demonstrate a novel role for TLR4.

An increased number of eosinophils in the esophagus is common in several esophageal and systemic diseases, and a prominent feature of eosinophilic esophagitis. Mouse models can provide insight into the mechanisms of eosinophil infiltration and their pathogenic role. SHARPIN-deficient cpdm mice develop a chronic proliferative dermatitis and an esophagitis characterized by epithelial hyperplasia and the accumulation of eosinophils in the serosa, submucosa, lamina propria and epithelium of the esophagus. We conducted a detailed investigation of the pathogenesis of the esophagitis by light microscopy, immunohistochemistry, and gene expression as the mice aged from 4 to 10 weeks. The thickness of the esophageal epithelium and the number of eosinophils in the esophagus both increased with age. There were scattered apoptotic epithelial cells in mice at 6 – 10 weeks of age that reacted with antibodies to activated caspase 3 and caspase 9. The expression of CCL11 (eotaxin-1), IL4, IL13 and TSLP was increased in cpdm mice compared with wild type (WT) mice, and there was no changein the expression of CCL24 (eotaxin-2), IL5 and IL33. The expression of chitinase-like 3 and 4 (YM1 and YM2) proteins, markers of type 2 inflammation, was greatly increased in cpdm mice, and this was replicated in vitro by incubation of WT esophagus in the presence of IL4 and IL13. Immunohistochemistry showed that these proteins were localized in esophageal epithelial cells. The severity of the esophagitis was not affected by crossing SHARPIN-deficient mice with lymphocyte-deficient Rag1 null mice indicating that the inflammation is independent of B and T lymphocytes.

Mammalian chitinase-like proteins belong to a family of proteins structurally related to chitinases but devoid of enzymatic activity. They have a postulated role in remodeling of extracellular matrix and defense mechanisms against chitin-containing pathogens. The expression of these proteins is increased in parasitic infections and allergic airway disease, but their expression in dermatitis has not been examined. The mRNA expression of two chitinase 3-like (Chi3L) proteins, Chi3L3 (Ym1) and Chi3L4 (Ym2), was determined in the skin of normal mice, chronic proliferative dermatitis (cpdm/cpdm) mutant mice and mice with experimentally induced contact hypersensitivity reaction. The localization of Chi3L3 and Chi3L4 proteins in cells was determined by fluorescence microscopy of double-labeled frozen sections of skin, and confirmed in vitro by stimulation of macrophages and mast cells with cytokines. Quantitative RT-PCR demonstrated a 976-fold increase of Chi3l4 mRNA expression and a 24-fold increase of Chi3l3 mRNA expression in the skin of cpdm/cpdm mice. Their expression was also increased in the ears of mice with 2,4-dinitrofluorobenzene-induced contact hypersensitivity, but the increase was greater for Chi3l3 mRNA (51-fold) than Chi3l4 mRNA (32-fold). Western blot analysis with an antibody against Chi3L3 and Chi3L4 confirmed the increased amount of these proteins in the skin of cpdm/cpdm mice. Two-color immunofluorescence identified macrophages, dendritic cells and mast cells as cellular sources of Chi3L3 and Chi3L4 proteins. Eosinophils and neutrophils did not contain detectable concentrations of these proteins. Treatment of macrophages and mast cells in vitro with interleukin-4 induced expression of Chi3l3 and Chi3l4 mRNA.

The evaluation of anti-aging intervention strategies in dogs would benefit from reliable quantitative biomarkers of aging. In the present study, the expression of various immune parameters was measured in young and old dogs to identify potential biomarkers of aging. The second goal of the study was to determine the effect of age on the immune response to vaccines. The immune function, including the antibody response to vaccines, was determined in 32 young adult (3.15+/-0.8 years of age) and 33 old dogs (12.1+/-1.3 years of age) of various breeds. Old dogs had a significantly lower lymphocyte proliferative response and a lower percentage of CD4+ T cells and CD45R+/CD4+ T cells, and a higher percentage of CD8+ T cells and a higher concentration of serum and salivary IgA. The most significant differences (P

Chronic proliferative dermatitis (cpdm) is a spontaneous mutation that results in eosinophilic inflammation in multiple tissues, including the skin. To determine the mechanisms underlying the eosinophilic inflammation, the expression of cytokines in the skin was determined. There was increased expression of IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor in the skin of cpdm/cpdm mice, and no change in IL-10 and TNF expression. Supernatants of cultured spleen cells of cpdm/cpdm mice contained an increased amount of IL-5 and IL-13, and a decreased amount of IFN-γ. The ability of the cpdm/cpdm mice to mount a delayed-type hypersensitivity response was greatly reduced. These data are consistent with impaired type 1 and excessive type 2 cytokine production in cpdm/cpdm mice. The significance of this imbalanced cytokine production was evident in the efficacy of systemic treatment of cpdm/cpdm mice with IL-12. Mutant mice treated for 3 weeks with IL-12 had minimal changes in the skin as opposed to the severe dermatitis in mice treated with the vehicle. Treatment with IL-11, which opposes the effect of IL-12, had no effect.

Chronic proliferative dermatitis (cpd) is a spontaneous mutation in C57BL/Ka mice (cpdm/cpdm). The dermatitis is characterized by redness, hairloss, scaling, pruritus and histologically by epithelial hyperproliferation, infiltration of eosinophils, macrophages and mast cells. Lesions similar to those in the skin occur in the esophagus and forestomach. In this paper, we describe the effect of drug treatments directed against epidermal hyperproliferation (calcipotriene and etretinate), against inflammation (corticosteroids and dapsone) and against pruritus (loratidine and capsaicin). The criteria used to objectively estimate the effect of the treatment were 1) macroscopic evaluation of the lesions (cpd score), 2) degree of epithelial hyperproliferation assessed by BrdU incorporation and epithelial thickness, and 3) microscopic evaluation of the inflammatory cells in the skin samples. Treatment of the cpdm/cpdm mice with calcipotriene (5 μg/day for 3 weeks) inhibited epidermal proliferation and the number of eosinophils. Systemic etretinate treatment (30 μg/g/day for 3 weeks) was not very effective. Topical corticosteroids (0.05 μg/day, for 3 weeks) exerted a therapeutic effect on the hyperproliferation and the number of eosinophils. Oral dapsone treatment (34 μg/g/day, for 5 weeks) reduced the BrdU incorporation in the skin and the epithelial thickness in the esophagus. The anti-histamine loratidine (orally, 1.7 μg/ g/day, for 4 weeks) reduced the severity of the lesions macroscopically, probably by suppressing the pruritus. Capsaicin (topically, 30 mM, for 5 weeks) also reduced the severity of the macroscopic observable lesions. Moreover, capsaicin reduced the dorsal and ventral epidermal thickness. The results from this and previous studies indicate that steroids (topically and systemically) and less strongly calcipotriene are the most effective treatments for the lesions observed in the cpdm/cpdm mice, since both hyperproliferation and the influx of eosinophils are reduced. Although the pathogenesis of the cpd lesions remains to be determined, our results indicate that the cpdm/cpdm mouse can be used to investigate new drugs for their possible application in chronic dermatitis.

Bovine tonsils are mucosal-associated lymphoid tissue (MALT) located at the entry of the pharynx where both inhaled and ingested antigens can induce an immune response. This study was conducted to determine the lymphocyte populations and adhesion molecule expression in the palatine tonsil (PT) and pharyngeal tonsil (PhT) of adult cattle and compare them with typical MALT (discrete Peyer's patches, PP) and a peripheral lymph node (parotid lymph node, PLN). The distribution of various lymphocyte subsets was determined in situ by immunofluorescence, and their proportions were determined by multicolor flow cytometry. The tonsils were similar to PP in the proportions of B- and T-cells (25-32% T-cells, 39-45% B-cells), and T cell subpopulations (CD4, CD8, and gammadelta). The PP contained the highest proportion of memory T-helper cells with beta7 integrin (30.3%+/-5.4), the tonsils intermediate (PT: 19.8%+/-4.4 and PhT: 19.7%+/-4.9), and the PLN had the lowest proportion (15.4%+/-3.1). The opposite relationship was observed with CD62L on naïve T- helper cells as PP had the lowest proportion (14.2%+/-6.4), the tonsils intermediate (PT: 17.4%+/-2.5 and PhT: 24.3%+/-7.3), and the PLN the highest proportion (45.3%+/-6.5). MAdCAM-1 was highly expressed in the high endothelial venules (HEV) of PP, with variable and weak expression in the tonsils and PLN. PNAd, on the other hand, was highly expressed in HEV of tonsils and PLN, and weakly expressed in the PP. These results indicate that the bovine tonsils share characteristics with both PP and PLN. The alpha4beta7/MadCAM-land CD62L/PNAd interaction may be involved in lymphocyte migration to the tonsils, but it is likely that other adhesion molecules participate as well. Similarities between the human and bovine tonsils suggest that cattle may provide a good model to study the role of the tonsil in the respiratory immune response.

Through careful mapping of the physiology of the T-zone and GC B-blast dynamics to a mathematical representation of the cell processes including proliferation, migration, differentiation, and cell death, a mathematical model is constructed to capture the dominant nominal primary, late follicular, and secondary humoral response toHaemophilus influenzaeType b. This model explicitly incorporates the dynamics of memory B-cells, T-zone and GC B-dynamics, IgM and IgG antibodies, avidity maturation, and IC presentation by FDCs into a coherent framework. This paper describes the relevant immunology, the pertinent physiological assumptions, the developed model, and the parameter identification procedure. The model parameters were found using a parameter identification procedure that capitalizes on the timing and interactions of certain dominant physiological attributes. Simulation results and validation tests indicate that the model reflects not only a nominal primary and secondary humoral immune response but also the tertiary and T-independent responses. The model shows robustness to variations in infection dosage, bacterial growth rate (virulence of the strain), and onset-timing of the secondary response. The utility of this model in studying the humoral immune response is demonstrated through suggested physiological assumptions, mechanisms, and rates to be eventually clinically evaluated as well as insights into vaccination design. The model and parameter identification techniques are easily adapted to other diseases which primarily evoke a humoral immune response.

Respiratory infectious diseases are an important cause of economic losses to the cattle industry. There is a need for an effective, easy to administer vaccine to the critical bacterial pathogens that cause pneumonia in cattle. An orally administered vaccine could be given to a large number of animals without significant stress to the animals and with minimal labor. The purpose of this study was to determine whether the oral administration of a model antigen (ovalbumin) in alginate microspheres could induce pulmonary immunity in cattle. Calves were vaccinated orally with ovalbumin (OVA) following either a subcutaneous (s.c.) or oral priming dose of OVA. Calves primed and boostered by oral administration (oral/oral) of OVA encapsulated in alginate microparticles had increased numbers of antigen-specific IgA ASCs (ASCs) in bronchoalveolar lavage (BAL) fluids. Calves that received a s.c. priming followed by an oral booster inoculation (s.c./oral) of OVA in alginate microspheres had a greater number of anti-OVA IgA, IgG1 and IgG2 ASCs in BALF. S.c./oral calves also had increased numbers of anti-OVA IgG1 ASCs in peripheral blood whereas oral/oral calves had none. S.c./oral calves had increased anti-OVA IgG1, IgG2, and IgA titers in BALF, and IgG1 and IgG2 in serum compared to both oral/oral and sham vaccinated calves. These results indicate that oral administration of antigen encapsulated in alginate microspheres results in a mucosal immune response in the respiratory tract of cattle. Furthermore, s.c. priming both enhanced the IgA response and stimulated an IgG1 and IgG2 response not seen in oral/oral calves. The difference in antibody isotype results suggest that design of the vaccination protocol can direct antibody responses as needed for a specific immunization program.

SummaryThe microRNA miR-155 has been implicated in regulating inflammatory responses and tumorigenesis, but its precise role in linking inflammation and cancer has remained elusive. Here, we identify a connection between miR-155 and Notch signaling in this context. Loss of Notch signaling in the bone marrow (BM) niche alters hematopoietic homeostasis and leads to lethal myeloproliferative-like disease. Mechanistically, Notch signaling represses miR-155 expression by promoting binding of RBPJ to the miR-155 promoter. Loss of Notch/RBPJ signaling upregulates miR-155 in BM endothelial cells, leading to miR-155-mediated targeting of the nuclear factor κB (NF-κB) inhibitor κB-Ras1, NF-κB activation, and increased proinflammatory cytokine production. Deletion of miR-155 in the stroma of RBPJ−/− mice prevented the development of myeloproliferative-like disease and cytokine induction. Analysis of BM from patients carrying myeloproliferative neoplasia also revealed elevated expression of miR-155. Thus, the Notch/miR-155/κB-Ras1/NF-κB axis regulates the inflammatory state of the BM niche and affects the development of myeloproliferative disorders.

Local immunization of the respiratory tract may be the best way to achieve protection against respiratory pathogens. In order to do so successfully, it is important to fully understand how the immune response to antigen administered via the respiratory route develops. We studied the respiratory and systemic immune response after subcutaneous (SC) and intrabronchial (IB) inoculation of calves with ovalbumin (OVA). Eight calves received two SC inoculations of OVA and eight other calves received two SC and three additional IB inoculations of OVA. The occurrence of OVA-specific antibodies and antibody-secreting cells (ASC) was measured over time using isotype-specific enzyme linked immunosorbent assay (ELISA) and ELISPOT. SC immunization of calves did not result in OVA-specific IgA in bronchoalveolar lavage (BAL) fluid. Subcutaneous priming followed by intrabronchial challenge caused an initial IgG1 response in the bronchoalveolar lavage fluid, followed by a large IgA response. The presence of IgG1-ASCs indicated that the IgG1 was at least partially locally produced. Most of the OVA-specific IgA in the BAL fluid was secreted by pulmonary ASCs as indicated by the large number of IgA-ASCs in BAL samples and the low serum level of OVA-specific IgA. Antigen-specific IgG1 ASCs were detectable among peripheral blood mononuclear cells after culture with OVA.

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 of TCR γδ + 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.

Nonhuman adenovirus (Ad) vectors derived from bovine Ad serotype 3 (BAd3) or porcine Ad serotype 3 (PAd3) can circumvent pre-existing immunity against human Ad (HAd). We have previously reported differential transduction of human and nonhuman cells by these Ad vectors, and their distinct receptor usage and biodistribution. To compare the induction of innate immunity, vector toxicity and vector uptake by Kupffer cells (KCs) following intravenous administration of PAd3, BAd3, or HAd5 vectors in mice, we determined mRNA expression levels of proinflammatory chemokines and cytokines, and Toll-like receptors (TLRs) in the liver and spleen. Tissue toxicity of these vectors was assessed by comparing serum levels of liver-specific enzymes, histopathology and Kupffer cell (KC) depletion. Compared to the HAd5 vector, PAd3 and BAd3 vectors were more potent stimulators of innate immune responses as indicated by enhanced mRNA expression of TLRs and proinflammatory chemokines and cytokine genes. Histopathological changes in the liver were most pronounced in HAd5-inoculated mice while BAd3- or PAd3-inoculated mice revealed mild histologic changes that were confined to early time points. Inoculation with HAd5 or PAd3 vectors resulted in a significant (P

Sharpin-deficient (Sharpin(cpdm)) mutant mice develop a chronic eosinophilic dermatitis. To determine the efficacy of eosinophil-depletion in chronic inflammation, Sharpin(cpdm) mice were treated with anti-IL5 antibodies. Mice treated with anti-IL5 had a 90% reduction of circulating eosinophils and a 50% decrease in cutaneous eosinophils after 10 days compared with sham-treated littermates. Reducing the number of eosinophils resulted in increased severity of alopecia and erythema and a significant increase in epidermal thickness. Skin homogenates from mice treated with anti-IL5 had decreased mRNA expression of arylsulfatase B (Arsb), diamine oxidase (amiloride-binding protein 1, also called histaminase; Abp1) and Il10, which are mediators that eosinophils may release to quench inflammation. Skin homogenates from mice treated with anti-IL5 also had decreased mRNA expression of Il4, Il5, Ccl11, kit ligand (Kitl) and Tgfa; and increased mRNA expression of Tgfb1, Mmp12 and tenascin C (Tnc). In order to further decrease the accumulation of eosinophils, Sharpin(cpdm) mice were crossed with IL5 null mice. Il5(-/-), Sharpin(cpdm)/Sharpin(cpdm) mice had a 98% reduction of circulating eosinophils and a 95% decrease in cutaneous eosinophils compared with IL5-sufficient Sharpin(cpdm) mice. The severity of the lesions was similar between IL5-sufficient and IL5-deficient mice. Double mutant mice had a significant decrease in Abp1, and a significant increase in Tgfb1, Mmp12 and Tnc mRNA compared with controls. These data indicate that eosinophils are not essential for the development of dermatitis in Sharpin(cpdm) mice and suggest that eosinophils have both pro-inflammatory and anti-inflammatory roles in the skin of these mice.

In vitro studies indicate that Cul4A ubiquitin ligases target for ubiquitin-mediated proteolysis regulators of cell-cycle progression, apoptosis, development, and DNA repair. In hematopoietic cell lines, studies by our group and others showed that Cul4A ligases regulate proliferation and differentiation in maturing myeloid and erythroid cells. In vivo, Cul4A-deficient embryos die in utero. Cul4A haploinsufficient mice are viable but have fewer erythroid and primitive myeloid progenitors. Yet, little more is known about Cul4A function in vivo. To examine Cul4A function in adults, we generated mice with interferon-inducible deletion of Cul4A. Cul4A deficiency resulted in DNA damage and apoptosis of rapidly dividing cells, and mutant mice died within 3 to 10 days after induction with dramatic atrophy of the intestinal villi, bone marrow, and spleen, and with hematopoietic failure. Cul4A deletion in vivo specifically increased cellular levels of the Cul4A ligase targets Cdt1 and p27Kip1 but not other known targets. Bone marrow transplantation studies with Cul4A deletion in engrafted cells specifically isolated analysis of Cul4A function to hematopoietic cells and resulted in hematopoietic failure. These recipients died within 9 to 11 days, demonstrating that in hematopoietic cells, Cul4A is essential for survival.

The dome epithelium of Peyer's patches from different parts of the intestine of four dogs was examined by scanning and transmission electron-microscopy and by alkaline phosphatase histochemistry on glycol-methacrylate embedded sections. M cells were scattered among more numerous enterocytes in duodenal and jejunal Peyer's patches, but constituted the major cellular component of dome epithelia of the ileal Peyer's patches. Alkaline phosphatase histochemistry demonstrated low enzyme activity in the brush border of M cells, as compared to enterocytes, in the duodenum and jejunum, allowing identification of M cells at the light microscopic level. Alkaline phosphatase activity was too low in ileal enterocytes to permit visualization of M cells. The presence of intrafollicular invaginations of dome epithelium is a consistent finding in duodenal Peyer's patches of the dog and these invaginations were characterized by few M cells, many intraepithelial lymphocytes and strong alkaline phosphatase activity.

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.

Nonhuman adenoviral (Ad) vectors have shown the potential to overcome the preexisting Ad immunity. In order to assess their utility for in vivo applications, it is important to evaluate biodistribution, induction of innate immune response, and toxicity of these vectors in a suitable animal model. In this study, FVB/n mice were inoculated via the intravenous route with porcine Ad type 3 (PAd3), bovine Ad type 3 (BAd3), or human Ad type 5 (HAd5) vector carrying the green fluorescent protein (GFP) gene as a reporter. Mice inoculated similarly with phosphate-buffered saline served as negative controls. Mice were sacrificed at 0.25, 0.5, 1, 2, 4, 8 and 16 days post-inoculation, and the blood, liver, spleen, kidneys, lungs and heart were collected for further analyses. Vector biodistribution and transgene expression were analyzed by real-time PCR and reverse-transcription PCR, respectively. The serum samples were analyzed for induction of inflammatory cytokines such as tumor necrosis factor (TNF) alpha (TNF-a), interleukin 6 (IL-6), and interferon gamma (IFN-g). Hepatotoxicity of Ad vectors was assessed by monitoring serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, and also by histopathology. At early time points (up to 2 days post-inoculation), the presence of PAd3 vector was detected in the lungs, spleen, and liver, and the BAd3 vector was detected in all tissues examined. Whereas, the presence of HAd5 vector was detected only in the liver suggesting that biodistribution of these nonhuman Ad vectors was considerably different than that of HAd5. Increased serum IL-6 levels were observed at 0.25 day post-inoculation in the PAd3-inoculated group, while increased IFN-g levels were seen on 8 days post-inoculation in the HAd5-inoculated group. Serum AST and ALT levels were elevated at early time points (up to 1day post-inoculation) in mice inoculated with either PAd3 or BAd3, but not in the HAd5-inoculated group. Surprisingly, elevated levels of these enzymes were noted at a late time point (8 days post-inoculation) only in the HAd5-inoculated group. Histopathologically, the PAd3 vector elicited mild hepatocellular apoptosis, and the HAd5 vector resulted in mild multifocal lymphocytic hepatitis, but no hepatotoxicity was observed in the BAd3-inoculated group. These results indicated that biodistribution, innate immune response and hepatotoxicity following intravenous inoculation of mice with PAd3, BAd3, or HAd5 vector were different. Further characterization of nonhuman Ad vectors will be valuable for designing improved Ad-based gene delivery vectors.

CXXC finger protein 1 (Cfp1), encoded by the Cxxc1 gene, binds to DNA sequences containing an unmethylated CpG dinucleotide and is an epigenetic regulator of both cytosine and histone methylation. Cxxc1-null mouse embryos fail to gastrulate, and Cxxc1-null embryonic stem cells are viable but cannot differentiate, suggesting that Cfp1 is required for chromatin remodeling associated with stem cell differentiation and embryogenesis. Mice homozygous for a conditional Cxxc1 deletion allele and carrying the inducible Mx1-Cre transgene were generated to assess Cfp1 function in adult animals. Induction of Cre expression in adult animals led to Cfp1 depletion in hematopoietic cells, a failure of hematopoiesis with a nearly complete loss of lineage-committed progenitors and mature cells, elevated levels of apoptosis, and death within two weeks. A similar pathology resulted following transplantation of conditional Cxxc1 bone marrow cells into wild type recipients, demonstrating this phenotype is intrinsic to Cfp1 function within bone marrow cells. Remarkably, the Lin- Sca-1+ c-Kit+ population of cells in the bone marrow, which is enriched for hematopoietic stem cells and multi-potential progenitor cells, persists and expands in the absence of Cfp1 during this time frame. Thus, Cfp1 is necessary for hematopoietic stem and multi-potential progenitor cell function and for the developmental potential of differentiating hematopoietic cells.

Chemokines direct the migration of leukocytes to sites of inflammation and are potential targets for anti-inflammatory therapy. Chronic proliferative dermatitis (cpdm/cpdm) mutant mice develop a persistent eosinophilic dermatitis associated with increased T H 2 cytokines in the skin. Expression patterns of chemokines in the skin of cpdm/cpdm mice were evaluated to define the mechanisms driving cutaneous infiltration by leukocytes. RNA isolated from the skin of mutant and littermate control mice revealed a significant increase in Ccl1 (TCA-3), Ccl2 (MCP-1), Ccl11 (eotaxin), Ccl17 (TARC), CxcllO (IP-10), and the chemokine receptor Ccr3. The concentration of CCL 11 protein was increased two- to threefold in the skin of cpdm/cpdm mice by enzyme-linked immunosorbent assay. In vitro culture of primary dermal fibroblasts from cpdm/cpdm and control mice with tumor necrosis factor, IL-4, and IL-13 stimulation did not reveal differences in their ability to secrete CCL11, suggesting that the increased chemokine expression observed in the skin of cpdm/cpdm mice is most likely caused by the increased T H 2 cytokines in the dermis of this mouse model. Treatment of cpdm/cpdm mice with CCL 11-neutralizing polyclonal antibodies did not affect the number of eosinophils in the skin or the severity of the dermatitis. Neutralizing multiple chemokines or chemokine receptors may be necessary to decrease eosinophil accumulation. The cpdm/cpdm mutant mouse is a potentially useful model to determine the role of various chemokines in eosinophil accumulation in chronic inflammation.

Background We have previously described microscopic and electron microscopic alterations in lymphoid organs of PCV2 inoculated mice as apoptosis. In this study we wanted to investigate the molecular pathogenetic mechanism of PCV2-induced apoptosis. Eight-week old BALB/c mice were either sham inoculated (control mice) or inoculated intraperitoneally (ip) and intranasally (in) with a single (sPCV mice) or multiple (mPCV mice) doses of PCV2. Four control mice and 4 sPCV mice were sacrificed 7, 14, 28 and 42 days post inoculation (PI). All 4 mPCV mice were sacrificed 42 days PI. Following necropsy, immunohistochemistry for caspase 3 and in-situ TUNEL assay were performed on sections of spleen, lymph nodes, thymus and ileum from control, sPCV and mPCV mice. In addition, total RNA was extracted from spleens of control, sPCV and mPCV mice for simultaneous detection and semiquantitation of bcl-2 homologues and various caspase mRNAs using a multiprobe RNase protection assay system. Results PCV2 replicated and was associated with apoptosis in spleens, lymph nodes and Peyer's patches of infected BALB/c mice. Upregulation of caspase 1, 2, 3, 6, 7, 8, 11 and 12 and upregulation for the transcripts of apoptosis inhibitors bcl-2, bcl-w and bcl-X and apoptosis promoters' bax, bak and bad was detected in spleens of sPCV and mPCV mice, but not control mice. Apoptosis was further confirmed by light and electron microscopic morphology as well as by positive TUNEL assay and detection of activated caspase 3. PCV2 nucleic acid was detected by in-situ hybridization in the nuclei and cytoplasm of such apoptotic cells. Conclusion The data presented here support the hypothesis that PCV2 induces apoptosis mediated through the activation of caspases 8 and 3 in the spleens of infected mice.

The goal of this project is to develop a real-time cell-based biosensor using Toll-like receptors (TLRs) for pathogen detection. Existing biosensors rely on technologies that recognize only specific target analytes, requiring prior knowledge of the possible contaminating agents. Innate immune cells express TLRs that recognize conserved pathogen-associated molecular patterns on bacteria, viruses, parasites and fungi. Using TLRs as the receptor element in this biosensor will eliminate the need for a priori knowledge of the threat. At least 10 different members of the TLR family are expressed on cells of the innate immune system, each responding to different attributes of pathogenic organisms. Through flow cytometry, TLRs 2, 4 and 9 have been identified on THP-1 cells, and TLRs 2, 3, 4, 5 and 9 have been detected on J774 cells. Western blotting has identified Erk activation upon lipopolysacharide (LPS), E. coli and Poly(I):(C) exposure in J774 cells, and upon LPS and E. coli exposure in THP-1 cells. Cellular model systems are being developed to distinguish between bacteria and virus by selective stimulation of TLR3 and TLR5 (TLR3 specifically recognizes double-stranded viral RNA and TLR5 detects bacterial flagellin). A target application of this technology is point-of-care diagnostics. Realtime detection of viruses in nasal or throat swabs could help decrease the inappropriate use of antibiotics.

Blood, spleen and liver of specific pathogen-free (SPF) cats and SPF cats experimentally infected with Bartonella henselae were examined. Using immunohistochemical labeling, no intracellular B. henselae were observed in tissues of any cats, but extracellular B. henselae were detected in tissues of infected cats. Pseudoinclusions were detected in erythrocytes of all cats using electron microscopy.

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.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gammac) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors and has a similar clinical phenotype to human XSCID. We have previously shown that the block in T-cell development is more profound in XSCID dogs than in genetically engineered gamma c-deficient mice. In this study we evaluated the B-cell function in XSCID dogs. In contrast to the marked decrease in peripheral B-cells in gamma c-deficient mice, XSCID dogs have increased proportions and numbers of peripheral B-cells as observed in XSCID boys. Canine XSCID B-cells do not proliferate following stimulation with the T-cell-dependent B-cell mitogen, pokeweed mitogen (PWM); however, they proliferate normally in response to the T-cell-independent B-cell mitogen, formalin-fixed, heat-killed Staphylococcus aureus. Canine XSCID B-cells are capable of producing IgM but are incapable of normal class-switching to IgG antibody production as demonstrated by in vitro stimulation with PWM and immunization with the T-cell-dependent antigen, bacteriophage PhiX174. Similar results have been reported for XSCID boys. Thus, it appears that gamma c-dependent cytokines have differing roles in human and canine B-cell development than in the mouse making the XSCID dog a valuable model for studying the role of these cytokines in B-cell development and function.

Peyer's patches (PP) are an important component in the immune response against intestinal pathogens. Two independent, spontaneous mutations in the mouse Sharpin gene (Sharpin(cpdm) and Sharpin(cpdm-Dem)) result in the absence of PP and disrupted splenic white pulp in adult mice, although a full complement of lymph nodes is present. Here we report that rudimentary PP begin to develop in Sharpin(cpdm) mice during embryogenesis, but lack the organizational patterns that are typical of this tissue. In the present study, small intestines examined at weekly intervals from birth to maturity showed spontaneous regression of PP in mutant mice with concurrent infiltration of granulocytes. At 5 to 6 weeks of age, only indistinct remnants of granulocytic accumulations remain. Transplantation of normal bone marrow into Sharpin(cpdm) mice at 7 days of age did not prevent regression of PP in bone marrow chimeras examined at 7 to 8 weeks of age. These findings indicate that SHARPIN expression is required for the normal development and maintenance, but not initiation, of PP.

Chronic proliferative dermatitis is a spontaneous mutation in C57BL/Ka mice ( cpdm/cpdm ) and is characterized by epithelial hyperproliferation, infiltration by eosinophils and macrophages, and vascular dilatation. To elucidate whether these pathologic features are the result of a local (skin) process or a consequence of a systemic disorder, transplantations were performed of full-thickness grafts of affected skin from cpdm/cpdm mice and normal skin from control (C57BL/Ka) mice on the back of cpdm/cpdm , C57BL/Ka and athymic nude mice. After 3 months, the grafts maintained the histologic phenotype of the donor animal. Intercellular adhesion molecule-1 continued to be expressed by basal keratinocytes of the cpdm/cpdm grafts after transplanation. In contrast the basal keratinocytes of the C57BL/Ka grafts onto cpdm/cpdm mice remained negative for intercellular adhesion molecule-1 3 months after transplantation. An increased number of proliferating keratinocytes was present in the cpdm/cpdm skin-graft transplanted to nudes or to C57BL/Ka mice based on short-term bromodeoxyuridine labeling. The bromodeoxyuridine incorporation in the keratinocytes of the control C57BL/Ka skin grafts transplanted to cpdm/cpdm , nude, or C57BL/Ka mice was the same as in the keratinocytes of normal C57BL/Ka mice. This study demonstrates that the pathological features found in the cpdm/cpdm mice are the result of a disorder in the epidermis or dermis and not due to systemic defect.

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 intimai 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.

C57BL/Ka mice with chronic proliferative dermatitis (cpdm/cpdm) develop chronic persistent skin lesions characterized by epidermal hyperplasia, infiltration by granulocytes and macrophages, and vascular dilatation. Similar lesions are present in other orthokeratotic epithelia in affected mice, in particular the esophagus and forestomach. Here, we report on further characterization of epidermal hyperplasia and the granulocytes. Keratinocytes of lesional skin, but not of normal skin, show round and electron-dense mitochondrial inclusions that are present in all layers of the epidermis. Similar inclusions are also present in the esophagus and forestomach of affected mice. There appears to be a direct relation between the presence of intramitochondrial inclusions and epidermal hyperplasia in the mouse. Furthermore, the presence of keratinocyte-derived apoptotic bodies in the epidermis, esophagus, and forestomach was frequently observed in the lesions, which is consistent with previous light microscopic observations of single cell death of keratinocytes. The granulocytes present in the skin, esophagus, and forestomach were mainly eosinophils. There were widespread gaps observed in the lamina densa in the epidermis that were mostly directly associated with dermal or epidermal eosinophils. This type of gap is also observed in psoriasiform diseases in humans. This electron microscopic study demonstrated that this mouse model should be useful to screen potential therapeutic strategies for psoriasiform and other inflammatory skin disorders.

This study describes the immunologic abnormalities during the acute phase of juvenile polyarteritis syndrome (JPS), a multisystem necrotizing vasculitis of young dogs with a predilection for the coronary arteries. JPS has striking clinical, laboratory, and pathologic similarities to Kawasaki disease (KD), the most common cause of acquired heart disease in children in the United States. The immunologic abnormalities include an increase in serum IgA, an increase in the percentage of peripheral B cells and a decrease in the percentage of total peripheral T cells, a marked suppression of the blastogenic response to mitogenic stimulation, an inability to generate immunoglobulin-secreting plasma cells following polyclonal activation, the presence of antineutrophil cytoplasmic antibodies, and evidence of monocyte/macrophage activation. These immunoregulatory abnormalities are similar to those observed in children during the acute phase of KD. This unique, naturally occurring animal model of necrotizing vasculitis may prove useful for investigating novel therapeutic interventions in the treatment of necrotizing vasculitis and may yield insight into the immunopathology and etiology of KD.

Murine intestinal intraepithelial lymphocytes (ilEL) represent a heterogeneous population of cells which contains both TCR alpha beta+ and TCR gamma delta+ cells. In contrast to the TCR alpha beta+ cell population in peripheral lymphoid organs, the TCR alpha beta+ ilEL contain high numbers of Thy-1- cells. This negativity of ilEL for Thy-1, a glycosylphosphatidyl inositol (GPI)-linked membrane protein, is not the result of conformational changes of the Thy-1 molecule. The absence of Thy-1 on TCR gamma delta+ and a subset of TCR alpha beta+ ilEL is furthermore not caused by a general defect in the processing of GPI-linked proteins, since Thy-1- ilEL mainly express another GPI-linked protein, Qa-2. However, TCR alpha beta+ and TCR gamma delta+ ilEL are also virtually negative for two other members of this family of activation-associated GPI-linked proteins, Ly-6A and Ly-6C. This unusual pattern of GPI-linked protein expression on TCR+ ilEL may be related to differences in cellular activation and signal transduction pathways between TCR+ ilEL and regular peripheral T cells.

Canine Peyer's patches were examined by light microscopy and immunohistochemistry for possible variations depending on the location within the small intestine and for similarities and dissimilarities to PPs from other species. The duodenal and jejunal PPs were characterized by relatively large domes and interfollicular areas. In contrast, the ileal PP had small domes and poorly developed interfollicular areas and very large follicles. T cells were found in the interfollicular area and corona and in lesser numbers in the dome and germinal centers. The ileal PP contained far fewer T cells than the proximal PPs. Domes of canine PPs contained some cytoplasmic IgA+ (cIgA+) and many cIgG+ cells. Peanut agglutinin (PNA) stained germinal center cells in a selective but not-uniform way and did not stain T cells.

Critical regulators of hematopoiesis are controlled by ubiquitin-mediated proteolysis. Cul4A encodes a core subunit of one ubiquitin ligase, and previous results with hematopoietic cell lines and with Cul4A haploinsufficient mice indicate that Cul4A is required for hematopoietic stem cell function and to maintain the homeostasis of progenitors, precursors, and mature hematopoietic cells. Because Cul4A -deficiency is embryonic lethal, we generated Cul4A conditional knockout mice to examine the requirement of Cul4A for hematopoiesis in adult mice. A mutant Cul4A allele ( Cul4A flox ) was constructed where its first coding exon was flanked by loxP sites. Transgenic mice with this mutant allele and the interferon-inducible Cre transgene, Mx1-Cre , were derived. When deletion of Cul4A was induced in Cul4A flox/flox Mx1-Cre mice, the animals died within 3–10 days of the beginning of induction. Necropsies performed four days after the beginning of induction showed that all of the tissues where Mx1-Cre was reported to be expressed appeared normal, except the bone marrow, spleen, and small intestine. The red pulp in the spleen was diminished, there were many fewer nucleated cells in the bone marrow, and the microvilli of the small intestine (duodenum) were dramatically shortened. The mass and total cellularity of mutant spleens were half of controls ( Cul4A flox/flox mice without Mx1-Cre ), and bone marrow total cellularity was one-tenth of controls. The frequency of mutant hematopoietic progenitors was reduced 3800-fold in the bone marrow and 80-fold in the spleen. Peripheral blood counts of mature myeloid and lymphoid cells were also dramatically reduced. To separate the in vivo effects of Cul4A -deficiency in hematopoietic cells from those in other cell types, conditional mutant bone marrow was transplanted into wild type recipients, these cells were allowed to engraft for 2 months, and then Cul4A deletion was induced. Mutant animals died within 9–11 days of the beginning of induction with bone marrow nearly empty of cells, spleens only 29% the mass of controls, myeloid and lymphoid counts in the peripheral blood reduced to nearly zero, hematocrits at only 21% of controls, and platelet counts at only 10% of controls. The small intestine, however appeared normal, indicating that Cul4A -deficiency in hematopoietic cells is sufficient to cause death. To examine the fate of Cul4A -deficient hematopoietic cells, deletion was induced in vivo in Cul4A flox/flox Mx1-Cre and control mice, and then bone marrow was harvested and cultured in vitro. Apoptotic cells were detected (either Annexin V positive, 7-AAD negative or TUNEL positive cells) 2–5 days after induction. At 4 and 5 days after induction, the frequency of apoptotic mutant cells was significantly greater than controls (P=0.01 and 0.03, respectively), and at 5 days the frequency of TUNEL positive cells was 4.5-fold greater in the mutant cells. Together, these results indicate that Cul4A -deficiency in hematopoietic cells results in apoptosis, a failure of the hematopoietic system, and death. Analyses of how the expression levels of Cul4A target proteins are altered by Cul4A -deficiency will be presented.

In 1928, Carlens reported on his detailed investigations of the morphology and ontogeny of lymphoid tissue in the intestinal tract of large domestic animals (1). He observed that the morphology and development of the ileal Peyer’s patch (PP) was different from the PP in the rest of the small intestine. Recent studies in sheep (2), cattle (3) and swine (4) have confirmed and expanded Carlens’ observations. The ileal PP has been shown to be functionally different from the jejunal PP and may represent the bursa-equivalent in sheep and cattle (3,5). In contrast, morphologic studies in mice (6), rats (7) and humans (8) do not indicate the existence of these regional differences of PP.

We have propagated concanavalin A-stimulated cells from canine Peyer's patches in vitro in the presence of interleukin-2 (IL-2). The cells were characterized as T cells by determination of their phenotype and by functional assays. They are IL-2 dependent and respond to IL-2 of murine, primate and canine origin. The long-term cultured cells provided help for immunoglobulin production by purified autologous B cells and suppressed IgG production by nonseparated autologous peripheral blood mononuclear cells.

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 gastro-intestinal 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.