Jeffrey R. Abbott

A Kaposi's Sarcoma-Associated Herpesvirus-Encoded Ortholog of MicroRNA miR-155 Induces Human Splenic B-Cell Expansion in NOD/LtSz-scid IL2Rγnull Mice

ABSTRACT MicroRNAs (miRNAs) are small noncoding RNA molecules that function as posttranscriptional regulators of gene expression. Kaposis sarcoma (KS)-associated herpesvirus (KSHV), a B-cell-tropic virus associated with KS and B-cell lymphomas, encodes 12 miRNA genes that are highly expressed in these tumor cells. One viral miRNA, miR-K12-11, shares 100% seed sequence homology with hsa-miR-155, an oncogenic human miRNA that functions as a key regulator of hematopoiesis and B-cell differentiation. So far, in vitro studies have shown that both miRNAs can regulate a common set of cellular target genes, suggesting that miR-K12-11 may mimic miR-155 function. To comparatively study miR-K12-11 and miR-155 function in vivo, we used a foamy virus vector to express the miRNAs in human hematopoietic progenitors and performed immune reconstitutions in NOD/LtSz-scid IL2Rγnull mice. We found that ectopic expression of miR-K12-11 or miR-155 leads to a significant expansion of the CD19+ B-cell population in the spleen. Subsequent quantitative PCR analyses of these splenic B cells revealed that C/EBPβ, a transcriptional regulator of interleukin-6 that is linked to B-cell lymphoproliferative disorders, is downregulated when either miR-K12-11 or miR-155 is ectopically expressed. In addition, inhibition of miR-K12-11 function using antagomirs in KSHV-infected human primary effusion lymphoma B cells resulted in derepression of C/EBPβ transcript levels. This in vivo study validates miR-K12-11 as a functional ortholog of miR-155 in the context of hematopoiesis and suggests a novel mechanism by which KSHV miR-K12-11 induces splenic B-cell expansion and potentially KSHV-associated lymphomagenesis by targeting C/EBPβ.

Feline immunodeficiency virus model for designing HIV/AIDS vaccines.

Feline immunodeficiency virus (FIV) discovered in 1986 is a lentivirus that causes AIDS in domestic cats. FIV is classified into five subtypes (A-E), and all subtypes and circulating intersubtype recombinants have been identified throughout the world. A commercial FIV vaccine, consisting of inactivated subtype-A and -D viruses (Fel-O-Vax FIV, Fort Dodge Animal Health), was released in the United States in 2002. The United States Department of Agriculture approved the commercial release of Fel-O-Vax FIV based on two efficacy trials using 105 laboratory cats and a major safety trial performed on 689 pet cats. The prototype and commercial FIV vaccines had broad prophylactic efficacy against global FIV subtypes and circulating intersubtype recombinants. The mechanisms of cross-subtype efficacy are attributed to FIV-specific T-cell immunity. Findings from these studies are being used to define the prophylactic epitopes needed for an HIV-1 vaccine for humans.

Caveolin 1 inhibits HIV replication by transcriptional repression mediated through NF-κB.

ABSTRACT Caveolin 1 (Cav-1), the scaffold protein of a specific membrane lipid raft called caveolae, has been reported to suppress HIV-1 replication. However, the mechanism by which Cav-1 inhibits HIV replication remains unclear. In this study, we investigated the mechanism by which Cav-1 inhibits HIV replication at the level of gene expression. Our results show that Cav-1 represses viral gene expression and that this suppression involves the NF-κB pathway. We used several approaches in different cell types, including primary CD4+ T cells and macrophages, to demonstrate the role of nuclear factor κB (NF-κB) in Cav-1-mediated inhibition of viral expression. A mutational analysis of the cis-acting element shows that the two NF-κB sites in the U3 region of the long terminal repeat (LTR) are critical for Cav-1-mediated inhibition of viral expression. In the presence of Cav-1, phosphorylation of IKKβ, IKKα, IκBα, and NF-κB p65 is dramatically reduced, while viral gene expression is suppressed. In addition, translocation of NF-κB p65 to the nucleus decreases substantially in the presence of Cav-1. Furthermore, significant inhibition of NF-κB activation and binding to target DNA are evident in the presence of Cav-1. These results establish evidence that Cav-1 inhibits HIV replication by transcriptional repression of viral gene expression and contributes to HIVs persistent infection of macrophages.

Colonic Immune Stimulation by Targeted Oral Vaccine

Background Currently, sufficient data exist to support the use of lactobacilli as candidates for the development of new oral targeted vaccines. To this end, we have previously shown that Lactobacillus gasseri expressing the protective antigen (PA) component of anthrax toxin genetically fused to a dendritic cell (DC)-binding peptide (DCpep) induced efficacious humoral and T cell-mediated immune responses against Bacillus anthracis Sterne challenge. Methodology/Principal Finding In the present study, we investigated the effects of a dose dependent treatment of mice with L. gasseri expressing the PA-DCpep fusion protein on intestinal and systemic immune responses and confirmed its safety. Treatment of mice with different doses of L. gasseri expressing PA-DCpep stimulated colonic immune responses, resulting in the activation of innate immune cells, including dendritic cells, which induced robust Th1, Th17, CD4+Foxp3+ and CD8+Foxp3+ T cell immune responses. Notably, high doses of L. gasseri expressing PA-DCpep (1012 CFU) were not toxic to the mice. Treatment of mice with L. gasseri expressing PA-DCpep triggered phenotypic maturation and the release of proinflammatory cytokines by dendritic cells and macrophages. Moreover, treatment of mice with L. gasseri expressing PA-DCpep enhanced antibody immune responses, including IgA, IgG1, IgG2b, IgG2c and IgG3. L. gasseri expressing PA-DCpep also increased the gene expression of numerous pattern recognition receptors, including Toll-like receptors, C-type lectin receptors and NOD-like receptors. Conclusion/Significance These findings suggest that L. gasseri expressing PA-DCpep has substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration and may be used as a safe oral vaccine against anthrax challenge.

Fungal Keratitis in a Gopher Tortoise (Gopherus polyphemus)

Abstract A free-ranging gopher tortoise (Gopherus polyphemus) presented for trauma and blindness. Fibrinous exudate obscured visualization of the globes. This exudative crust extended from the conjunctival fornices through the palpebral fissure and was manually removed. Ophthalmic examination revealed bilateral corneal ulcerations and scarring and phthisis bulbi of the left globe. Histology of the crust revealed a necrotic conjunctivitis with intralesional fungal hyphae. Culture of the corneal ulcer of the left eye isolated moderate growth of a mixed fungal flora consisting of Curvularia sp. and Aspergillus sp. Miconazole ophthalmic solution was administered and the ulcers in both eyes healed, but corneal edema continued. After 2 mo of treatment with miconazole, tramadol, acetylcysteine, hypertonic saline ointment, artificial tears, and hypertonic saline flushes, the right eye was normal with only a small scar. The left eye remained phthisical. This is the first report of fungal keratitis in a wild reptile and a gopher tortoise.

Mucosal injury and inflammatory cells in response to brief ischaemia and reperfusion in the equine large colon.

REASON FOR PERFORMING STUDY Intestinal ischaemia and reperfusion (I/R) can activate inflammatory cells in the equine colon, although effects on different types of inflammatory cells have received little attention. OBJECTIVES To assess early mucosal injury, the reaction of mucosal neutrophils, eosinophils, mast cells and macrophages, and cyclooxygenase (COX)-1 and -2 expression in response to I/R in the equine large colon. METHODS Large colon ischaemia was induced for 1 h (1hI) followed by 4 h of reperfusion in 6 horses, and mucosal biopsies were sampled before and after ischaemia, and after 1, 2 and 4 h of reperfusion. Semithin sections (500 nm) of epon-embedded biopsies were stained with toluidine blue for histomorphometric evaluation. The number and distribution of mucosal macrophages (CD163), neutrophils (calprotectin), eosinophils (LUNA) and mast cells (toluidine blue) were determined, and mucosal COX-1 and -2 expression was identified. RESULTS Ischaemia caused epithelial cell and nuclear swelling (mean ± s.e. nuclear width; control: 2.7 ± 0.2 µm vs. 1hI: 4.2 ± 0.2 µm; P<0.01), subepithelial oedema (control: 0.2 ± 0.1 µm vs. 1hI: 3.2 ± 0.2 µm; P<0.01) and increased epithelial apoptosis (control: 14.3 ± 4.1 apoptotic cells/mm mucosa vs. 1hI: 60.4 ± 14.0 apoptotic cells/mm mucosa; P<0.01). COX-2 expression (P<0.01) was evident after ischaemia. Reperfusion caused paracellular fluid accumulation (control: 0.9 ± 0.1 µm vs. 1hI: 0.6 ± 0.6 µm vs. 1hI + 4hR: 1.6 ± 0.2 µm; P<0.05). Epithelial repair started at 1 h of reperfusion (P<0.001), followed by migration of neutrophils into the mucosa after 2 h (control: 72.3 ± 18.4 cells/mm(2) mucosa vs. 1hI + 2hR: 1149.9 ± 220.6 cells/mm(2) mucosa; P<0.01). Mucosal eosinophils, mast cells and macrophages did not increase in numbers but were activated. CONCLUSIONS Epithelial injury and COX-2 expression caused by short-term hypoxia were followed by intense inflammation associated with epithelial repair during reperfusion. POTENTIAL RELEVANCE Equine colonic mucosa subjected to a brief period of ischaemia can repair during reperfusion, despite increased mucosal inflammation.

Utilization of Feline ELISPOT for Mapping Vaccine Epitopes

A commercial feline immunodeficiency virus (FIV) vaccine consisting of inactivated dual-subtype viruses was released in the USA in 2002 and released subsequently over the next 6 years in Canada, Australia, New Zealand, and Japan. Based on the genetic, morphologic, and biochemical similarities between FIV and human immunodeficiency virus-1 (HIV-1), FIV infection of domestic cats is being used as a small animal model of HIV/AIDS vaccine. Studies on prototype and commercial FIV vaccines provide new insights to the types of immunity and the vaccine epitopes required for an effective human HIV-1 vaccine. ELISPOT assays to detect cytokines, chemokines, and cytolytic mediators are widely used to measure the magnitude and the types of cellular immunity produced by vaccination. Moreover, such approach has identified regions on both HIV-1 and FIV proteins that induce robust antiviral cellular immunity in infected hosts. Using the same strategy, cats immunized with prototype and commercial FIV vaccines are being analyzed by feline interferon-γ and IL-2 ELISPOT systems to identify the vaccine epitope repertoire for prophylaxis.

Evolutionarily conserved T-cell epitopes on FIV for designing an HIV/AIDS vaccine.

This review will discuss the current state of the human HIV-1 vaccine trials including the safety consideration of vaccine composition and difficulties in determining and defining protective immunity and epitopes to HIV-1. Vaccines in animal models of lentivirus infection are compared. In particular, the findings from the prototype FIV vaccine and the HIV-1 protein immunizations studies in cats are discussed, as well as the resulting research regarding a potential HIV-1 vaccine design based on evolutionarily conserved T-cell epitopes.

Cranial mediastinal liposarcoma in a horse.

A 23-year-old Anglo-Arabian mare was presented with tachypnea, dyspnea, and pitting edema of the ventral thoracic subcutis. On necropsy, a tan to red, friable, irregularly shaped mass (23 × 20 × 18 cm) occupied the cranial mediastinum. Histologically, the mass was classified as a liposarcoma and was composed of short interlacing bundles of spindle-shaped to irregularly rounded cells with discrete, variably sized, clear cytoplasmic vacuoles, which were stained with oil red O in frozen sections of formalin-fixed tissue.

Chordomas at High Prevalence in the Captive Population of the Endangered Perdido Key Beach Mouse (Peromyscus polionotus trissyllepsis)

The Perdido Key beach mouse (Peromyscus polionotus trissyllepsis) is a critically endangered subspecies of the oldfield mouse. The captive population, currently maintained by 3 Florida zoos, is entirely derived from just 3 wild-caught ancestor mice. Necropsy and histopathology revealed chordoma of the vertebral column in 38 of 88 (43%) mice. The tumors were locally expansile and invasive masses of large physaliferous (vacuolated) cells with small, round, hyperchromatic nuclei, similar to the “classic” form of chordomas described in humans. Primary tumors rarely contained small amounts of bone and cartilaginous matrix, characteristic of the “chondroid” form. Neoplastic cells contained abundant granules positive by the periodic acid–Schiff reaction. Brachyury and cytokeratin AE1/AE3 were detected in neoplastic cells by immunohistochemistry, but uncoupling protein 1 was not identified. Primary tumors occurred along the entire vertebral column—cervical, 5 of 38 (13%); thoracic, 16 (42%); lumbar, 13 (34%); and sacral, 10 (26%)—and 10 (26%) mice had multiple primary masses. Metastases to the lungs were noted in 13 of the 38 (34%) mice. Mice diagnosed with chordomas postmortem ranged from 424 to 2170 days old, with a mean of 1399 days. The prevalence of chordoma was not significantly different between males (n = 23 of 50; 46%) and females (n = 15 of 38; 39%).