Search

The phosphate content of commercial ovalbumin was increased from 1.8 to 3.2 mol PO(4)/mol ovalbumin by conjugation of phosphoserine and reduced to 1.2 or 0.14 mol PO(4)/mol ovalbumin by treatment with potato acid phosphatase. The four ovalbumin samples were completely adsorbed by aluminum hydroxide adjuvant due to electrostatic attraction of the negatively charged ovalbumin and the positively charged aluminum hydroxide adjuvant as well as by ligand exchange of phosphate groups with surface hydroxyl groups. Elution from aluminum hydroxide adjuvant upon exposure to interstitial fluid was inversely related to the degree of phosphorylation of the ovalbumin. The ovalbumin sample containing 3.2 mol PO(4)/mol ovalbumin did not elute while the ovalbumin sample containing 0.14 mol PO(4)/mol ovalbumin eluted completely from aluminum hydroxide adjuvant during exposure to interstitial fluid for 30 min. Adsorption of the four ovalbumin samples by aluminum phosphate adjuvant was directly related to the degree of phosphorylation of ovalbumin. Adsorption was due to ligand exchange as an electrostatic repulsive force operated between the negatively charged ovalbumin samples and the negatively charged aluminum phosphate adjuvant. The potential for ligand exchange decreased as the phosphorylation of ovalbumin decreased. Elution upon exposure to interstitial fluid was inversely related to the degree of phosphorylation and was more extensive than observed for aluminum hydroxide adjuvant. Adsorption of ovalbumin by aluminum-containing adjuvants and elution upon exposure to interstitial fluid can be controlled by the degree of phosphorylation of both ovalbumin and the aluminum-containing adjuvant.

An important step in the induction of an immune response to vaccines is the internalization of antigens by antigen presenting cells, such as dendritic cells (DCs). Many current vaccines are formulated with antigens adsorbed to an aluminum-containing adjuvant. Following injection of the vaccine the antigens may either elute or stay adsorbed to the adjuvant surface. Antigens, which elute from the adjuvant surface, are internalized by dendritic cells through macropinocytosis while those that remain adsorbed are internalized with the adjuvant particle by phagocytosis. The relative efficiency of these two routes of internalization was studied. Alpha casein (AC) labeled with a green fluorescent dye was selected as the model antigen. In order to model vaccine antigens that elute from aluminum-containing adjuvants following administration, dendritic cells were incubated with a solution of fluorochrome-labeled alpha casein. To model vaccine antigens that do not elute from aluminum-containing adjuvants following administration, dendritic cells were exposed to fluorochrome-labeled alpha casein adsorbed to aluminum hydroxide adjuvant (AH). Alpha casein has eight phosphate groups and adsorbs to aluminum hydroxide adjuvant through ligand exchange. Alpha casein does not elute from aluminum hydroxide adjuvant upon exposure to cell culture media. The uptake of antigen by dendritic cells was determined at 0.5, 1, 2 and 3h by confocal microscopy and flow cytometry. Dendritic cells internalized both alpha casein in solution and alpha casein adsorbed to aluminum hydroxide adjuvant. However, the mean fluorescence intensity of dendritic cells incubated with adsorbed alpha casein was four times greater than dendritic cells incubated with alpha casein in solution. In addition, the internalization of alpha casein was enhanced when the mean aggregate diameter of the adjuvant in the cell culture media was reduced from 17 microm to 3 microm. It was concluded that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and the aggregate size of the adjuvant was smaller than dendritic cells which are approximately 10 microm in diameter.

Differential gene expression between the metastatic human colon cancer cell line HT29p and its nonmetastatic counterpart HT29-MTX was revealed by suppression subtractive hybridization. Fifty-eight individual genes showed increased mRNA levels in HT29p cells. Only 15 of these genes had been related to transformation in previous studies; the majority of genes are new candidates encoding proteins relevant for the metastatic process. Cancer profiling arrays as well as in situ hybridization study revealed that at least some of the genes obtained in the SSH screen are also differentially expressed in human tumors.

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

The effect of the degree of adsorption after exposure to interstitial fluid on the immune response in mice to model vaccines containing ovalbumin, alpha casein or dephosphorylated alpha casein adsorbed to aluminum hydroxide adjuvant was studied. Ovalbumin and dephosphorylated alpha casein were adsorbed in the vaccine but were completely eluted when exposed to interstitial fluid for 4 h. The presence of aluminum hydroxide adjuvant in the vaccine produced immunopotentiation compared to a solution of the protein even though the protein desorbed rapidly upon subcutaneous administration. In contrast, alpha casein was completely adsorbed to aluminum hydroxide adjuvant in both the vaccine and upon exposure to interstitial fluid. Immunopotentiation by aluminum hydroxide adjuvant was also observed in this model vaccine compared to a solution of alpha casein. The results indicated that antigen presenting cells can take up desorbed antigen from interstitial fluid as well as antigen adsorbed to aluminum-containing adjuvants.

Aluminum hydroxide adjuvant was pretreated with six concentrations of potassium dihydrogen phosphate to produce a series of adjuvants with various degrees of phosphate substitution for surface hydroxyl. The adsorption of three phosphorylated proteins (alpha casein, dephosphorylated alpha casein, and ovalbumin) by the phosphate-treated aluminum hydroxide adjuvants was studied. The phosphorylated proteins were adsorbed by ligand exchange of phosphate for hydroxyl even when an electrostatic repulsive force was present. However, the extent (adsorptive capacity) and strength (adsorptive coefficient) of adsorption was inversely related to the degree of phosphate substitution of the aluminum hydroxide adjuvant. Exposure of vaccines containing aluminum hydroxide adjuvant and phosphorylated antigens to phosphate ion in the formulation or during manufacture should be minimized to produce maximum adsorption of the antigen.

Caroline G. McPhee1, F. Jason Duncan2, Kathleen A. Silva1, Lloyd E. King Jr.3, Harm HogenEsch4, Derry C. Roopenian1, Helen B. Everts2, and John P. Sundberg1,3 1The Jackson Laboratory, Bar Harbor, ME 2Department of Nutrition, The Ohio State University, Columbus, OH 3Division of Dermatology, Vanderbilt University, Nashville, TN 4Department of Comparative Pathobiology, Purdue University, West Lafayette, IN, Bar Harbor, Maine, USA

Pasteurella multocida is an important bacterial pathogen of domestic rabbits. To evaluate the ability of a thiocyanate extract (PTE) of P. multocida to stimulate an immune response and protect against infection with P. multocida, rabbits were immunized subcutaneously or intranasally on Days 7, 21 and 35. Cholera toxin, a potent mucosal adjuvant, was included in one treatment group. Rabbits immunized subcutaneously (SC) or intranasally (IN) had significant increases in serum anti-PTE IgG but not IgA. In contrast, only rabbits immunized IN with PTE developed significant titers of nasal lavage anti-PTE IgA and cholera toxin significantly enhanced this response. In a second study rabbits were immunized via the drinking water with PTE incorporated into alginate microparticles on Days 7, 14 and 21. Mild increases in serum IgG were noted in rabbits immunized with PTE in microparticles, with or without cholera toxin, and this increase was significant (Por=0.05) on Day 21 for rabbits receiving PTE and cholera toxin. Nasal lavage anti-PTE IgA was significantly (Por=0.05) increased in rabbits immunized orally with PTE, with or without cholera toxin, in microparticles. This effect was not enhanced by cholera toxin. Rabbits orally immunized with PTE in microparticles had significantly fewer colony forming units of homologous P. multocida recovered from the lungs and nasopharynx following an intranasal challenge. These results demonstrate that PTE incorporated into alginate microparticles and administered orally is immunogenic and confers protective immunity.

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 (P0.001) 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 (P0.05) 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.

Objective—To determine whether routine vaccination induces antibodies against bovine thyroglobulin and autoantibodies against canine thyroglobulin in dogs. Design—Prospective study. Animals—20 healthy research Beagles and 16 healthy pet dogs. Procedure—For the research Beagles, 5 dogs were vaccinated with a multivalent vaccine and a rabies vaccine, 5 dogs received only the multivalent vaccine, 5 dogs received only the rabies vaccine, and 5 dogs were unvaccinated controls. The multivalent vaccine was administered at 8, 10, 12, 16, 20, 26, and 52 weeks of age and every 6 months thereafter. The rabies vaccine was administered at 16 and 52 weeks of age and then once per year. Blood was collected from all dogs at 8, 16, and 26 weeks of age and then 4 times yearly. Assays for antibodies directed against bovine and canine thyroglobulin were performed prior to and 2 weeks after each yearly vaccination. For the pet dogs, blood was collected prior to and 2 weeks after 1 vaccination. Results—In the research Beagles, there was a significant increase in anti-bovine thyroglobulin antibodies in all vaccinated dogs, compared with control dogs. There was a significant increase in anti-canine thyroglobulin antibodies in the 2 groups of dogs that received the rabies vaccine but not in the group that received the multivalent vaccine alone. In the pet dogs, there was a significant increase in anti-canine thyroglobulin antibodies after vaccination but no significant change in anti-bovine thyroglobulin antibodies. Conclusions and Clinical Relevance—Recent vaccination may result in increased anti-canine thyroglobulin antibodies. Whether these antibodies have a deleterious effect on canine thyroid function is unknown. (J Am Vet Med Assoc 2002;221:515–521)

Aluminum adjuvants are widely used in human and veterinary vaccines. They are appropriate adjuvants for vaccines that confer protection by inducing antibodies via the induction of a type 2 immune response, but they do not induce cytotoxic T cell and cell-mediated immunity. The mechanisms by which aluminum adjuvants selectively enhance the immune response are poorly understood. Following exposure to interstitial fluid in vitro and in vivo, most antigens are rapidly desorbed from aluminum adjuvants, suggesting that sustained release of antigen from a depot does not significantly contribute to the adjuvant effect of aluminum compounds. However, the adsorption of antigens onto aluminum salts may result in a high local concentration of antigen at the injection site and enhance the uptake by antigen-presenting cells. Aluminum compounds can further enhance the immune response by direct or indirect stimulation of dendritic cells, activation of complement and by inducing the release of chemokines. The relative importance of these mechanisms remains to be determined.

SHARPIN is a component of the linear ubiquitination assembly complex and a key regulator of NFkB and integrin signaling. SHARPIN-deficient mice develop a phenotype known as chronic proliferative dermatitis (cpdm), characterized by progressive epidermal hyperplasia, apoptosis of keratinocytes, cutaneous and systemic eosinophilic inflammation, and hypoplasia of secondary lymphoid organs. We recently reported that the cutaneous inflammation in SHARPIN-deficient mice (Sharpincpdm) develops independently of B and T lymphocytes. We therefore sought to determine the role of innate lymphoid cells (ILCs) in the dermatitis of Sharpincpdm mice. ILCs were identified as a discrete population of CD45+ Lin−CD90.2hi cells in the skin and draining lymph nodes (LN) of wild type (WT) and Sharpincpdm mice. The number of ILCs was markedly increased in Sharpincpdm mice. The majority of cells were group 2 ILC (ILC2) as indicated by labeling for GATA3, IL5, and IL13. The skin of Sharpincpdm mice had increased expression of Il33 and Tslp mRNA. To determine the role of IL33, double mutant mice were generated in which the receptor for IL33 (Il1lr1 also known as ST2) was deleted. Loss of IL33-signaling greatly reduced the number of ILCs and reduced the severity of the dermatitis. These experiments suggest that the dermatitis in Sharpincpdm mice is driven by IL33-dependent ILC2. Supported by the NIH (AR049288).

Diseases resulting from infection by group A streptococcus (GAS) are an increasing burden on global health. A novel vaccine was developed targeting infection by Streptococcus pyogenes. The vaccine incorporates a recombinant fusion protein antigen (SpeAB) which was engineered by combining inactive mutant forms of streptococcal pyrogenic exotoxin A (SpeA) and streptococcal pyrogenic exotoxin B (SpeB) from S. pyogenes. A rational, scientific approach to vaccine development was utilized to determine optimal formulation conditions with aluminum adjuvants. Investigations of the pH stability profile of SpeAB concluded the antigen was most stable near pH 8. Incorporation of the stabilizers sucrose and mannitol significantly enhanced the stability of the antigen. Vaccines were formulated in which most of the SpeAB was adsorbed to the adjuvant or remained in solution. A SpeAB vaccine formulation, stabilized with sucrose, in which the antigen remains adsorbed to the aluminum adjuvant retained the greatest potency as determined by evaluation of neutralizing antibody responses in mice. This vaccine has great potential to provide a safe and effective method for prevention of GAS disease.

SHARPIN is a key regulator of NFKB and integrin signaling. Mice lacking Sharpin develop a phenotype known as chronic proliferative dermatitis (CPDM), typified by progressive epidermal hyperplasia, apoptosis of keratinocytes, cutaneous and systemic eosinophilic inflammation, and hypoplasia of secondary lymphoid organs. Rag1(-/-) mice, which lack mature B and T cells, were crossed with Sharpin(-/-) mice to examine the role of lymphocytes in CDPM. Although inflammation in the lungs, liver, and joints was reduced in these double mutant mice, dermatitis was not reduced in the absence of functional lymphocytes, suggesting that lymphocytes are not primary drivers of the inflammation in the skin. Type 2 cytokine expression is increased in CPDM. In an attempt to reduce this aspect of the phenotype, Il4ra(-/-) mice, unresponsive to both IL4 and IL13, were crossed with Sharpin(-/-) mice. Double homozygous Sharpin(-/-) , Il4ra(-/-) mice developed an exacerbated granulocytic dermatitis, acute system inflammation, as well as hepatic necrosis and mineralization. High expression of CHI3L4, normally seen in CPDM skin, was abolished in Sharpin(-/-) , Il4ra(-/-) double mutant mice indicating the crucial role of IL4 and IL13 in the expression of this protein. Cutaneous eosinophilia persisted in Sharpin(-/-) , Il4ra(-/-) mice, although expression of Il5 mRNA was reduced and the expression of Ccl11 and Ccl24 was completely abolished. TSLP and IL33 were both increased in the skin of Sharpin(-/-) mice and this was maintained in Sharpin(-/-) , Il4ra(-/-) mice suggesting a role for TSLP and IL33 in the eosinophilic dermatitis in SHARPIN-deficient mice. These studies indicate that cutaneous inflammation in SHARPIN-deficient mice is autoinflammatory in nature developing independently of B and T lymphocytes, while the systemic inflammation seen in CPDM has a strong lymphocyte-dependent component. Both the cutaneous and systemic inflammation is enhanced by loss of IL4 and IL13 signaling indicating that these cytokines normally play an anti-inflammatory role in SHARPIN-deficient mice.

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.

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.

Tumor necrosis factor-α (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.

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.

Summary The chronic proliferative dermatitis (cpdm) mouse mutation was mapped to mouse Chromosome 15 using an intraspecific cross between C57BL/KaLawRij cpdm/cpdm and BALB/cJ mice. A second autosomal recessive mutation that resulted in a phenotype similar to that of cpdm arose spontaneously in a colony of OcB-3/Dem recombinant congenic mice. This new mutation was found to be allelic with cpdm. Therefore, the gene symbol for the allelic mutation is cpdmDem. The phenotype of these mutant mice consists of eosinophil-driven severe and progressive inflammatory changes in multiple organs including the skin and lungs.

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.

The purpose of this study was to determine whether neonatal cats develop and maintain a persistent bacteremia for longer than do adult cats with a normal mature immune system, and whether neonatal cats are susceptible to infection with Bartonella henselae by oral inoculation. Neonatal specific pathogen-free (SPF) cats were inoculated with B. henselae intradermally ( n = 4) or orally ( n = 5) or with 0.9% NaCl ( n = 2). Blood was collected periodically through 16 weeks post-inoculation (PI) for serology, bacteriology and complete blood count. Cats inoculated orally or intradermally at 3–5 days of age were bacteremic through 12–16 weeks PI, similar to what is documented for adult cats inoculated intradermally or intravenously. One cat inoculated at age 2 weeks was bacteremic through 10 weeks PI; the other was not bacteremic. Intradermally inoculated neonatal cats produced serum IgG antibodies to B. henselae but orally inoculated neonatal cats did not. Infected cats with and without serum IgG antibodies to B. henselae became blood-culture negative simultaneously, suggesting that IgG is not required to clear bacteremia.

Urocanic acid (2-propenoic acid, 3-[ lH-imidazol-4(5)-yl], UA)? is one of the smallest molecules to have stimulated global interest among biologists, environmentalists, photochemists, photobiologists, medicinal chemists and immunologists. Its history can be traced back more than a century to when it was first found in the urine of dogs (1). Interest in the molecule remained dormant until the middle of the century but rekindled in the late 1940s when it was detected in animal skin and sweat. This led to the proposal that UA acts as a natural sunscreen, possibly as a specific photoprotecting agent for DNA because of the overlap of the UA and DNA absorption spectra (2). Since 1983 there has been an explosive growth in UA research, primarily as a consequence of the proposal by De Fabo and Noonan (3) that the cis photoisomer (cUA) of trans-UA (tUA) could be responsible for the phenomenon of photoimmunosuppression. In a recent literature survey we found that about 25 research papers have appeared every year over the last decade involving UA. Several comprehensive reviews of the molecule's photobiology, photochemistry and photophysics have appeared (4

Most infectious diseases begin at a mucosal surface. Prevention of infection must therefore consider ways to enhance local immunity to prevent the attachment and invasion of microbes. Despite this understanding, most vaccines depend on parenterally administered vaccines that induce a circulating immune response that often does not cross to mucosal sites. Administration of vaccines to mucosal sites induces local immunity. To be effective requires that antigen be administered often. This is not always practical depending on the site where protection is needed, nor comfortable to the patient. Not all mucosal sites have inductive lymphoid tissue present as well. Oral administration is easy to do, is well accepted by humans and animals and targets the largest inductive lymphoid tissue in the body in the intestine. Oral administration of antigen requires protection of antigen from the enzymes and pH of the stomach. Polymeric delivery systems are under investigation to deliver vaccines to the intestine while protecting them from adverse conditions that could adversely affect the antigens. They also can enhance delivery of antigen specifically to the inductive lymphoid tissue. Sodium alginate is a readily available, inexpensive polymer that can be used to encapsulate a wide variety of antigens under mild conditions. Orally administered alginate microspheres containing antigen have successfully induced immunity in mice to enteric (rotavirus) pathogens and in the respiratory tract in cattle with a model antigen (ovalbumin). This delivery system offers a safe, effective means of orally vaccinating large numbers of animals (and perhaps humans) to a variety of infectious agents.

The expression of keratins and filaggrin by keratinocytes is a highly regulated process and depends on their state of differentiation and proliferation. As such, these proteins can be used as markers to determine if keratinocyte differentiation is normal. Mutant cpdm/cpdm mice develop a chronic skin disease characterized by epidermal hyperplasia and inflammation. Immunohistochemical staining for the basal keratins K5 and K14 revealed expression in the basal and suprabasal cell layers. The expression of K1 and K10 was reduced and limited to the outer layers of the stratum spinosum. Keratin 6 was expressed in the suprabasal layers of affected skin, and throughout all layers in severely affected skin. Filaggrin was present in the stratum granulosum which had variable thickness. These results indicate that the differentiation of keratinocytes in cpdm/cpdm mice was normal. The altered distribution and expression of keratins in comparison with the skin of control mice was the result of hyperproliferation.

Five female specific pathogen-free (SPF) cats inoculated intradermally with B. henselae and bacteremic for 4 weeks, and one cat inoculated with 0.9% NaCl, were bred with uninfected SPF male cats. The uninfected female became pregnant with one breeding, while three infected cats became pregnant 1-12 weeks later, after repeated breedings. Two infected females either did not become pregnant or maintain pregnancies despite repeated breedings. Infected cats produced anti-B. henselae IgM and IgG antibodies. Fetuses and kittens of infected cats were not infected and did not produce anti-B. henselae antibodies. Male cats bred with infected females did not become infected or seroconvert. Maternal anti-B. henselae IgG antibodies detected in sera of kittens 2 weeks post-partum were no longer detectable 10 weeks post-partum. These findings suggest that B. henselae causes reproductive failure in female cats, but is not transmitted transplacentally, in colostrum or milk, or venereally. Infected cats immunosuppressed with methylprednisolone acetate after their kittens were weaned had no detectable bacteria in tissues, suggesting that they were no longer infected.

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.

Aluminum-containing adjuvants are widely used in human and veterinary vaccines, but their mechanism of action is not well understood. Recent evidence suggests an important role for inflammation in the immune response to aluminum-adjuvanted vaccines. To better understand this process, vaccines with aluminum adjuvant were injected into naïve or previously immunized mice and the injection sites were characterized for the corresponding primary and secondary inflammatory response at different time points after immunization. Inflammatory cells appeared at the injection site between 2h and 6h after vaccination, dominated by neutrophils at first, followed by macrophages, and later eosinophils and MHCII(+) cells. The number of cells at the injection site increased over time, except neutrophils, which decreased in number after day 2. There was extensive phagocytosis of aluminum adjuvant particles by macrophages. In secondary immunized mice, a faster and more robust recruitment of eosinophils, macrophages, and antigen presenting cells was observed at the injection site. The enhanced recruitment of inflammatory cells in previously immunized mice coincided with increased expression of relevant chemokines at the injection site. Since neutrophils accumulated first in response to aluminum-adjuvanted vaccines, their role was evaluated by depleting them prior to vaccination. Neutrophil depletion transiently reduced the recruitment of macrophages but it did not change the recruitment of eosinophils and MHCII(+) cells or the quality and magnitude of the antibody response.

Aluminum-containing adjuvants are widely used in preventive vaccines against infectious diseases and in preparations for allergy immunotherapy. The mechanism by which they enhance the immune response remains poorly understood. Aluminum adjuvants selectively stimulate a Th2 immune response upon injection of mice and a mixed response in human beings. They support activation of CD8 T cells, but these cells do not undergo terminal differentiation to cytotoxic T cells. Adsorption of antigens to aluminum adjuvants enhances the immune response by facilitating phagocytosis and slowing the diffusion of antigens from the injection site which allows time for inflammatory cells to accumulate. The adsorptive strength is important as high affinity interactions interfere with the immune response. Adsorption can also affect the physical and chemical stability of antigens. Aluminum adjuvants activate dendritic cells via direct and indirect mechanisms. Phagocytosis of aluminum adjuvants followed by disruption of the phagolysosome activates NLRP3-inflammasomes resulting in the release of active IL-1β and IL-18. Aluminum adjuvants also activate dendritic cells by binding to membrane lipid rafts. Injection of aluminum-adjuvanted vaccines causes the release of uric acid, DNA, and ATP from damaged cells which in turn activate dendritic cells. The use of aluminum adjuvant is limited by weak stimulation of cell-mediated immunity. This can be enhanced by addition of other immunomodulatory molecules. Adsorption of these molecules is determined by the same mechanisms that control adsorption of antigens and can affect the efficacy of such combination adjuvants. The widespread use of aluminum adjuvants can be attributed in part to the excellent safety record based on a 70-year history of use. They cause local inflammation at the injection site, but also reduce the severity of systemic and local reactions by binding biologically active molecules in vaccines.

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.

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 E-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 10-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 CB7BL/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.

Oral vaccination is a simple, efficient way of inducing immunity at mucosal surfaces. The slow development of oral vaccines has been mainly due to the lack of suitable delivery systems. We have used hydrogel microspheres to deliver various vaccines to several animal species by oral administration. Oral delivery of vaccines using alginate microspheres elicited the production of secretory IgA (sIgA) at the mucosal surfaces in mice, rabbits, and cattle. Oral vaccination of chicken resulted in an increased delayed-type hypersensitivity, a cell-mediated immune response, indicating a positive response to the vaccine. Our studies have clearly shown that alginate microspheres are effective for the oral administration of vaccines.

Mice with spontaneous mutations in the Sharpin gene develop chronic proliferative dermatitis that is characterized by eosinophilic inflammation of the skin and other organs with increased expression of type 2 cytokines and dysregulated development of lymphoid tissues. The mutant mice share phenotypic features with human hypereosinophilic syndromes. The biological function of SHARPIN and how its absence leads to such a complex inflammatory phenotype in mice are poorly understood. However, recent studies identified SHARPIN as a novel modulator of immune and inflammatory responses. The emerging mechanistic model suggests that SHARPIN functions as an important adaptor component of the linear ubiquitin chain assembly complex that modulates activation of NF-κB signalling pathway, thereby regulating cell survival and apoptosis, cytokine production and development of lymphoid tissues. In this review, we will summarize the current understanding of the ubiquitin-dependent regulatory mechanisms involved in NF-κB signalling, and incorporate the recently obtained molecular insights of SHARPIN into this pathway. Recent studies identified SHARPIN as an inhibitor of β1-integrin activation and signalling, and this may be another mechanism by which SHARPIN regulates inflammation. Furthermore, the disrupted lymphoid organogenesis in SHARPIN-deficient mice suggests that SHARPIN-mediated NF-κB regulation is important for de novo development of lymphoid tissues.

Spontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases: Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines. The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive. Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity. Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen. In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86. However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide. Mutant BMDC had defective activation of NF-κB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact. A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naïve CD4(+) T cells. In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response.

Background Streptococcus pneumoniae causes widespread morbidity and mortality. Current vaccines contain free polysaccharides or protein-polysaccharide conjugates, and do not induce protection against serotypes that are not included in the vaccines. An affordable and broadly protective vaccine is very desirable. The goal of this study was to determine the optimal formulation of a killed whole cell pneumococcal vaccine with aluminum-containing adjuvants for intramuscular injection. Methods Four aluminium-containing adjuvants were prepared with different levels of surface phosphate groups resulting in different adsorptive capacities and affinities for the vaccine antigens. Mice were immunized three times and the antigen-specific antibody titers and IL-17 responses in blood were analyzed. Results Although all adjuvants induced significantly higher antibody titers than antigen without adjuvant, the vaccine containing aluminum phosphate adjuvant (AP) produced the highest antibody response when low doses of antigen were used. Aluminum hydroxide adjuvant (AH) induced an equal or better antibody response at high doses compared with AP. Vaccines formulated with AH, but not with AP, induced an IL-17 response. The vaccine formulated with AH was stable and retained full immunogenicity when stored at 4°C for 4 months. Conclusions Antibodies are important for protection against systemic streptococcal disease and IL-17 is critical in the prevention of nasopharyngeal colonization by S. pneumoniae in the mouse model. The formulation of the whole killed bacterial cells with AH resulted in a stable vaccine that induced both antibodies and an IL-17 response. These experiments underscore the importance of formulation studies with aluminium containing adjuvants for the development of stable and effective vaccines.

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

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.

The relationship between depot formation and immunopotentiation was studied by comparing the retention of antigen at the inoculation site with antibody production in rats. A model (111)In-labeled alpha casein (IDCAS) antigen was formulated into four vaccines: IDCAS adsorbed onto either aluminum hydroxide adjuvant (AH) or aluminum phosphate adjuvant (AP); non-adsorbed IDCAS with phosphate-treated AP (PTAP); and IDCAS solution. Gamma scintigraphy showed the order of retention following subcutaneous administration to be: AH adsorbed>AP adsorbed>non-adsorbed with PTAP=solution. The antibody titers followed the order: non-adsorbed with PTAP=AP adsorbed>AH adsorbed>>solution. The presence of an aluminum-containing adjuvant was essential for immunopotentiation, but retention of the antigen at the inoculation site was not required.

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.

Ageing is associated with a decline in functional competence of the immune system, sometimes referred to as immunosenescence. As this increases the susceptibility of dogs to infectious diseases, it is important to determine if the efficacy of vaccines is affected by ageing. Studies to date suggest that the primary response to vaccines may be compromised in old dogs, but recall responses remain intact. Information on the effect of ageing on the duration of protective immunity following vaccination is needed.

The excellence of a journal depends on the quality of peer review. The process of publishing scientific discoveries depends on considerable work by unpaid experts who volunteer time as peer reviewers. Peer reviewers are the essential gatekeepers who maintain high standards of scientific rigor. Equally important, many manuscripts are significantly improved when the authors respond to insightful suggestions from peer reviewers. Veterinary Pathology has been fortunate over the years in having a pool of highly dedicated and high-quality reviewers. Scientists are motivated to spend time reviewing manuscripts for a number of reasons. Altruism and service to the community of science, and to the discipline of veterinary pathology, motivate many to spend time reading and reviewing manuscripts, but these are not the only reasons. Curiosity and the desire to stay current are equally important. Reviewers enjoy the challenge involved in the careful analysis and assessment of a manuscript. Finally, for the success of Veterinary Pathology, manuscripts must be reviewed carefully and rapidly, and this would be impossible without the active participation of the broad scientific community. Conflict with other workloads is a major reason that potential reviewers decline invitations to review manuscripts. As discussed in the 2007 American College of Veterinary Pathologists (ACVP) Town Hall meeting, pathologists have increasing demands on their time, and the stresses of multitasking continue to increase. The problem is compounded by the retirement or death of some of the stalwart experts of veterinary pathology.

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.