Dalen W. Agnew

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

Cystic Endometrial Hyperplasia in Elephants

Most captive female elephants are nulliparous and aged and many have endometrial disease, factors that may hinder fertility. This study characterized the pathologic features and demographic distribution of endometrial lesions from 27 captive Asian (Elephas maximus) and 13 African elephants (Loxodonta africanus), 12- to 57-years of age. The principal lesion was marked cystic and polypoid endometrial hyperplasia (CEH), present in 67% of Asian and 15% of African elephants ranging from 26 to 57 years. The lower prevalence in African elephants likely reflects their younger age range in this study. Fourteen of 15 affected elephants with breeding information were nulliparous. These results suggest that CEH and polyps are common in aged nulliparous elephants, and the severity of these lesions may impair fertility. These findings will be useful in the interpretation of ultrasonographic findings during reproductive examinations of potential breeding cows. Also, breeding programs should focus on younger animals.

First analysis of the secretome of the canine heartworm, Dirofilaria immitis

BackgroundThe characterization of proteins released from filariae is an important step in addressing many of the needs in the diagnosis and treatment of these clinically important parasites, as well as contributing to a clearer understanding of their biology. This report describes findings on the proteins released during in vitro cultivation of adult Dirofilaria immitis , the causative agent of canine and feline heartworm disease. Differences in protein secretion among nematodes in vivo may relate to the ecological niche of each parasite and the pathological changes that they induce.MethodsThe proteins in the secretions of cultured adult worms were run on Tris-Glycine gels, bands separated and peptides from each band analysed by ultra mass spectrometry and compared with a FastA dataset of predicted tryptic peptides derived from a genome sequence of D. immitis.ResultsThis study identified 110 proteins. Of these proteins, 52 were unique to D. immitis . A total of 23 (44%) were recognized as proteins likely to be secreted. Although these proteins were unique, the motifs were conserved compared with proteins secreted by other nematodes.ConclusionThe present data indicate that D. immitis secretes proteins that are unique to this species, when compared with Brugia malayi. The two major functional groups of molecules represented were those representing cellular and of metabolic processes. Unique proteins might be important for maintaining an infection in the host environment, intimately involved in the pathogenesis of disease and may also provide new tools for the diagnosis of heartworm infection.

Real-Time Polymerase Chain Reaction Testing for the Detection of Mycobacterium genavense and Mycobacterium avium Complex Species in Avian Samples

Abstract SUMMARY. Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR. In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan® PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan® PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products. The TaqMan® PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan® PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan® PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan® PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan® PCR products. The TaqMan® PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation.

Uterine Mast Cells and Immunoglobulin-E Antibody Responses During Clearance of Tritrichomonas foetus

We showed earlier that Tritrichomonas foetus-specific bovine immunoglobulin (Ig)G1 and IgA antibodies in uterine and vaginal secretions are correlated with clearance of this sexually transmitted infection. Eosinophils have been noted in previous studies of bovine trichomoniasis but the role of mast cells and IgE responses have not been reported. The hypothesis that IgE and mast cell degranulation play a role in clearance was tested in 25 virgin heifers inseminated experimentally and infected intravaginally with T. foetus strain D1 at estrus and cultured weekly. Groups were euthanatized at 3, 6, 9, or 12 weeks, when tissues were fixed and secretions were collected for culture and antibody analysis. Immunohistochemistry using a monoclonal antibody to a soluble lipophosphoglycan (LPG)-containing surface antigen (TF1.17) demonstrated antigen uptake by uterine epithelial cells. Lymphoid nodules were detected below antigen-positive epithelium. Little IgG2 antibody was detected but IgG1, IgA, IgM, and IgE T. foetus-specific antibodies increased in uterine secretions at weeks 6 and 9 after infection. This was inversely proportional to subepithelial mast cells numbers and most animals cleared the infection by the sampling time after the lowest mast cell count. Furthermore, soluble antigen was found in uterine epithelium above inductive sites (lymphoid nodules). Cross-linking of IgE on mast cells by antigen and perhaps LPG triggering appears to have resulted in degranulation. Released cytokines may account for production of predominantly Th2 (IgG1 and IgE) and IgA antibody responses, which are related to clearance of the infection.

Analysis of a Survey Database of Pet Food-Induced Poisoning in North America

Following the outbreak of pet food-induced nephrotoxicity in March 2007, a voluntary online survey of all AAVLD-accredited laboratories, commercial laboratories, and veterinary clinics across North America was conducted. There was no information on toxicity of melamine or factors affecting the disease outcome following exposure to melamine in pets. Data were collected from affected pets to learn about the disease outcome and the affected pet population. The web-based electronic survey used the online tool, SurveyMonkey™. Data were collected between April 5 and October 31, 2007. Four hundred fifty-one cases of 586 reported cases met the criteria for inclusion in the study. Most reported cases were from California, Texas, Michigan, Florida, and Ontario. Of the 451 cases, 424 were reported as affected. Of these, 278 cases (65.6%) were cats and 146 (34.4%) were dogs. A total of 278 pets (171 cats and 107 dogs) were reported to have died (a ratio of 1.6:1). However, within species, there was a higher percentage of deceased dogs (73.3%) than cats (61.5%). Of the affected pet population, older male cats with preexisting disease conditions were more likely to be deceased. Analysis of the pets in this large database of naturally affected pets yielded interesting findings. It showed that more cats than dogs were affected and also that preexisting renal diseases and old age predicted the most severe outcome (death or euthanasia) than any other factors.

Malignant Lymphoma in African Lions (Panthera Leo)

Malignant lymphoma has become an increasingly recognized problem in African lions (Panthera leo). Eleven African lions (9 male and 2 female) with clinical signs and gross and microscopic lesions of malignant lymphoma were evaluated in this study. All animals were older adults, ranging in age from 14 to 19 years. Immunohistochemically, 10 of the 11 lions had T-cell lymphomas (CD3+, CD79a–), and 1 lion was diagnosed with a B-cell lymphoma (CD3–, CD79a+). The spleen appeared to be the primary site of neoplastic growth in all T-cell lymphomas, with involvement of the liver (6/11) and regional lymph nodes (5/11) also commonly observed. The B-cell lymphoma affected the peripheral lymph nodes, liver, and spleen. According to the current veterinary and human World Health Organization classification of hematopoietic neoplasms, T-cell lymphoma subtypes included peripheral T-cell lymphoma (4/11), precursor (acute) T-cell lymphoblastic lymphoma/leukemia (2/11), chronic T-cell lymphocytic lymphoma/leukemia (3/11), and T-zone lymphoma (1/11). The single B-cell lymphoma subtype was consistent with diffuse large B-cell lymphoma. Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) testing by immunohistochemistry on sections of malignant lymphoma was negative for all 11 lions. One lion was seropositive for FeLV. In contrast to domestic and exotic cats, in which B-cell lymphomas are more common than T-cell lymphomas, African lions in this study had malignant lymphomas that were primarily of T-cell origin. Neither FeLV nor FIV, important causes of malignant lymphoma in domestic cats, seems to be significant in the pathogenesis of malignant lymphoma in African lions.

Surgical Amputation of a Digit and Vacuum-Assisted-Closure (V.A.C.) Management in a Case of Osteomyelitis and Wound Care in an Eastern Black Rhinoceros (Diceros bicornis michaeli)

Abstract:  A 14-yr-old female eastern black rhinoceros (Diceros bicornis michaeli) presented with progressive suppurative osteomyelitis in her left hind lateral toe. β-Hemolytic Streptococcus sp. was isolated. The animal was treated with multiple systemic antibiotics, and topical wound cleansing. Repeated debridements and nail trimmings were performed for 5 mo prior to electing amputation. The toe was surgically amputated under general anesthesia between the first and second phalanges. Analgesia was diffused into the wound topically via a catheter and elastomeric pump. The open amputation site was covered with adherent drapes and a negative-pressure wound therapy device provided vacuum-assisted closure (V.A.C.®) for 72 hr. Three months later this animal developed a deep dermal ulcer on the lateral aspect of the right hind limb, at the level of the stifle. Methicillin-resistant Staphylococcus aureus was isolated. The wound was managed by initial daily lavage, followed by 1 mo of V.A.C. therapy, with 72 hr between dressing changes. Clinically, this therapy expedited the formation of healthy granulation tissue and overall healing was accelerated. The animal tolerated the machine and bandage changes well via operant conditioning. The use of negative-pressure wound therapy appeared to shorten time to resolution of slow-healing wounds in black rhinoceros.

Intrauterine Group A Streptococcal Infections Are Exacerbated by Prostaglandin E2

Streptococcus pyogenes (Group A Streptococcus; GAS) is a major cause of severe postpartum sepsis, a re-emerging cause of maternal morbidity and mortality worldwide. Immunological alterations occur during pregnancy to promote maternofetal tolerance, which may increase the risk for puerperal infection. PGE2 is an immunomodulatory lipid that regulates maternofetal tolerance, parturition, and innate immunity. The extent to which PGE2 regulates host immune responses to GAS infections in the context of endometritis is unknown. To address this, both an in vivo mouse intrauterine (i.u.) GAS infection model and an in vitro human macrophage–GAS interaction model were used. In C57BL/6 mice, i.u. GAS inoculation resulted in local and systemic inflammatory responses and triggered extensive changes in the expression of eicosanoid pathway genes. The i.u. administration of PGE2 increased the mortality of infected mice, suppressed local IL-6 and IL-17A levels, enhanced neutrophilic inflammation, reduced uterine macrophage populations, and increased bacterial dissemination. A role for endogenous PGE2 in the modulation of antistreptococcal host defense was suggested, because mice lacking the genes encoding the microsomal PGE2 synthase-1 or the EP2 receptor were protected from death, as were mice treated with the EP4 receptor antagonist, GW627368X. PGE2 also regulated GAS–macrophage interactions. In GAS-infected human THP-1 (macrophage-like) cells, PGE2 inhibited the production of MCP-1 and TNF-α while augmenting IL-10 expression. PGE2 also impaired the phagocytic ability of human placental macrophages, THP-1 cells, and mouse peritoneal macrophages in vitro. Exploring the targeted disruption of PGE2 synthesis and signaling to optimize existing antimicrobial therapies against GAS may be warranted.

Evaluation of hypoxia in a feline model of head and neck cancer using 64Cu-ATSM positron emission tomography/computed tomography

BackgroundHuman and feline head and neck squamous cell carcinoma (HNSCC) share histology, certain molecular features, as well as locally aggressive and highly recurrent clinical behavior. In human HNSCC, the presence of significant hypoxia within these tumors is considered an important factor in the development of a more aggressive phenotype and poor response to therapy. We hypothesized that feline head and neck tumors, particularly HNSCC, would exhibit hypoxia and that 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) positron emission tomography/computed tomography (PET/CT) would permit detection of intratumoral hypoxia.Methods12 cats with measureable head and neck tumors were given 64Cu-ATSM and iodinated contrast for PET/CT scan. The presence or absence of hypoxia was also assessed using an intratumoral fluorescent life-time probe to quantitate pO2 and pimonidazole immunohistochemical staining in biopsy specimens. In two cats, intratumoral O2 and 64Cu-ATSM uptake was measured before and after treatment with anti-angiogenic agents to determine the effect of these agents on hypoxia.ResultsEleven of twelve feline tumors demonstrated significant 64Cu-ATSM uptake, regardless of malignant or benign etiology. The presence (and absence) of hypoxia was confirmed using the fluorescent O2 detection probe in nine tumors, and using pimonidazole staining in three tumors. Squamous cell carcinomas (HNSCC) demonstrated the highest degree of hypoxia, with Tmax/M ratios ranging from 4.3 to 21.8. Additional non-neoplastic tissues exhibited 64Cu-ATSM uptake suggestive of hypoxia including reactive draining lymph nodes, non-malignant thyroid pathology, a tooth root abscess, and otitis media. In two cats with HNSCC that received anti-vascular agents, the pattern of 64Cu-ATSM uptake was altered after treatment, demonstrating the potential of the feline model to study the modulation of tumor oxygenation.ConclusionFeline HNSCC serves as a clinically relevant model for the investigation of intratumoral hypoxia including its measurement, modulation and targeting.