Ingeborg M. Langohr

Proposal of a 2-Tier Histologic Grading System for Canine Cutaneous Mast Cell Tumors to More Accurately Predict Biological Behavior

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy

Arterial tissues collected from Ossabaw swine bearing metabolic syndrome-induced cardiovascular plaques are characterized by multimodal nonlinear optical microscopy that allows coherent anti-Stokes Raman scattering, second-harmonic generation, and two-photon excitation fluorescence imaging on the same platform. Significant components of arterial walls and atherosclerotic lesions, including endothelial cells, extracellular lipid droplets, lipid-rich cells, low-density lipoprotein aggregates, collagen, and elastin are imaged without any labeling. Emission spectra of these components are obtained by nonlinear optical microspectrometry. The nonlinear optical contrast is compared with histology of the same sample. Multimodal nonlinear optical imaging of plaque composition also allows identification of atherosclerotic regions that are vulnerable to rupture risk. The demonstrated capability of nonlinear optical microscopy for label-free molecular imaging of atherosclerotic lesions with 3-D submicrometric resolution suggests its potential application to the diagnosis of atherosclerotic plaques, determination of their rupture risk, and design of individualized drug therapy based on plaque composition.

Imaging and Quantitative Analysis of Atherosclerotic Lesions by CARS-Based Multimodal Nonlinear Optical Microscopy

Objective—The purpose of this study was to assess the ability of label-free multimodal nonlinear optical (NLO) microscopy to characterize, and thus enable quantitative in situ analyses of, different atherosclerotic lesion types, according to the original scheme suggested by the AHA Committee. Methods and Results—Iliac arteries were taken from 24 male Ossabaw pigs divided into lean control and metabolic syndrome groups and were imaged by multimodal NLO microscopy where sum-frequency generation (SFG) and 2-photon excitation fluorescence (TPEF) were integrated on a coherent anti-Stokes Raman scattering (CARS) microscope platform. Foam cells, lipid deposits, matrices, and fibrous caps were visualized with submicron 3D resolution. Starting from the adaptive intimal thickening in the initial stage to the fibrous atheroma or mineralization in the advanced stages, lesions were visualized without labels. Histological staining of each lesion confirmed the lesion stages. Lipid and collagen contents were quantitatively analyzed based on the CARS and SFG signals. Lipid accumulation in thickened intima culminated in type IV whereas the highest collagen deposition was found in Type V lesions. Luminal CARS imaging showed the capability of viewing the location of superficial foam cells that indicate relatively active locus in a lesion artery. Conclusions—We have demonstrated the capability of CARS-based multimodal NLO microscopy to interrogate different stages of lesion development with subcellular detail to permit quantitative analysis of lipid and collagen contents.

Dietary Fish Oil Alters T Lymphocyte Cell Populations and Exacerbates Disease in a Mouse Model of Inflammatory Colitis

Inflammatory bowel diseases (IBD) increase the risk of developing colorectal cancer. Dietary components that reduce inflammation are associated with lower cancer risk. The long-chain omega-3 fatty acid docosahexaenoic acid (DHA) is present in fish oil and has potent anti-inflammatory properties. The objective of this study is to determine whether dietary fish oil enriched with DHA (DFO) could reduce experimentally induced colitis and colon cancer risk in a mouse model. When SMAD3-/- mice are exposed to Helicobacter hepaticus, mild colitis is observed 4 weeks postinfection. Mice were fed isocaloric diets modified to include corn oil, safflower oil, or DFO (doses ranging from 0.75% to 6.00%) as the fatty acid source for 8 weeks. Mice were gavaged with H. hepaticus; DFO feeding was continued; and mice were sacrificed 4 weeks after infection. The colon and cecum were collected for histopathology. Spleens and mesenteric lymph nodes were collected and analyzed for T-cell populations using flow cytometry. Contrary to expectations, DFO induced severe colitis and adenocarcinoma formation. DFO consumption was associated with decreased CD8(+) cell frequency and diminished CD69 expression on CD4(+) and CD8(+) T-cell populations. Mice consuming DFO also exhibited higher FoxP3(+) CD25(+) CD4(+) T regulatory cell frequency, FoxP3 expression, and altered L-selectin expression during infection. We concluded that DFO-fed mice may be less equipped to mount a successful response to H. hepaticus infection, increasing colon cancer risk. These results support the need to establish a tolerable upper limit for DHA intake particularly in the context of chronic inflammatory conditions such as IBD.

Current State of Knowledge on Porcine Circovirus Type 2-Associated Lesions

Porcine circovirus type 2 (PCV2), a small single-stranded DNA virus, was initially discovered in 1998 and is highly prevalent in the domestic pig population. Disease manifestations associated with PCV2 include postweaning multisystemic wasting disease (PMWS), enteric disease, respiratory disease, porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure. Although these clinical manifestations involve different organ systems, there is considerable overlap in clinical expression of disease and presence of lesions between pigs and within herds. It is now widely accepted that PCV2 can be further subdivided into different types, of which PCV2a and PCV2b are present worldwide and of greatest importance. This review will focus on PCV2-associated lesions in different organ systems.

Supplementation with Galacto-Oligosaccharides Increases the Percentage of NK Cells and Reduces Colitis Severity in Smad3-Deficient Mice

The gut microbiota plays an essential role in intestinal immunity. Prebiotics, including galacto-oligosaccharides (GOS), are fermentable fibers that beneficially affect the host by stimulating the growth of specific microbial populations. We investigated the effect of GOS on colitis development and on immune variables in Smad3-deficient mice treated with the pathogen Helicobacter hepaticus. Mice were supplemented daily with 5000 mg GOS/kg body weight 2 wk prior to infection and 4 wk postinfection, a time period during which colitis severity peaks in this model. Mice (n = 4-8/treatment at each time) were killed preinfection (0 d) and at 3, 7, and 28 d postinfection to evaluate immune variables in the spleen and in mesenteric lymph nodes (MsLN) by flow cytometry. Colon and cecum samples were collected for histopathologic analysis. Fecal pellets (n = 8-9/treatment) were collected prior to infection to measure relative changes in Bifidobacterium ssp. and Lactobacillum ssp. by real-time PCR. GOS significantly reduced colitis severity in response to H. hepaticus (P < 0.0001). This was associated with a significant increase in the percentage of NK cells in the spleen (P < 0.001) and in MsLN (P < 0.001) at 3 d postinfection and a 1.5-fold increase in fecal Bifidobacterium ssp. (P = 0.003). GOS stimulated NK expression of CCR9, a chemokine receptor involved in lymphocyte trafficking to the gut preinfection (0 d) in the blood (P = 0.02), spleen (P = 0.033), and MsLN (P = 0.017). In addition, GOS stimulated colonic IL-15 production 3 d postinfection (P < 0.001). These data suggest that GOS reduces colitis by modulating the function and trafficking of NK cells and may provide a novel therapeutic strategy for individuals with inflammatory bowel disease.

Pseudorabies virus infection in Oklahoma hunting dogs

Pseudorabies is caused by Suid herpesvirus 1, a member of the Alphaherpesvirinae subfamily. Although pigs are the natural host of Pseudorabies virus (PRV), the virus has a broad host range and may cause fatal encephalitis in many species. The United States obtained PRV-free status in 2004 after the virus was eradicated from domestic swineherds, but the virus is still present in feral swine populations. The current report describes PRV infection in 3 dogs that were used to hunt feral swine. The dogs developed clinical signs including facial pruritus with facial abrasions, dyspnea, vomiting, diarrhea, ataxia, muscle stiffness, and death. Two were euthanized, and 1 died within approximately 48 hr after onset of clinical signs. The salient histologic changes consisted of neutrophilic trigeminal ganglioneuritis with neuronophagia and equivocal intranuclear inclusion bodies. Pseudorabies virus was isolated from fresh tissues from 2 of the dogs, and immunohistochemistry detected the virus in the third dog. Virus sequencing and phylogeny, based upon available GenBank sequences, revealed that the virus was likely a field strain that was closely related to a cluster of PRV strains previously identified in Illinois. Though eradicated from domestic swine in the United States, PRV is present in populations of feral swine, and should therefore continue to be considered a possible cause of disease in dogs and other domestic animals with compatible clinical history and signs. Continued surveillance is necessary to prevent reintroduction of PRV into domestic swine.

Congenital Neosporosis in Goats from the State of Minas Gerais, Brazil

Congenital Neospora caninum infection was diagnosed in two Saanen goat kids from two distinct herds with a history of abortion and weak newborn goat kids in the Southern region of the State of Minas Gerais, Brazil. The first kid was weak at birth, had difficulty to rise and was unable to nurse. Gross lesions of porencephaly and hydrocephalus ex vacuo were seen. Multifocal necrosis, gliosis and non-supurative encephalitis were observed in the brain. Several parasitic cysts with a thick wall that reacted strongly only with polyclonal antiserum to Neospora caninum were seen in the cerebral cortex, brain stem and cerebellum. The second kid was born from a Neospora caninum seropositive mother that aborted in the last pregnancy. It was born without clinical signs. The diagnosis of neosporosis was based on antibody titer of 1:800 to N. caninum by indirect fluorescence antibody test obtained from blood collected before the goat kid ingested the colostrum and Neospora caninum DNA was detected by polymerase chain reaction and sequenced from placenta. This is the first report of neosporosis in goats in the southeast region of Brazil.

Comparative Effects of n-3, n-6 and n-9 Unsaturated Fatty Acid-Rich Diet Consumption on Lupus Nephritis, Autoantibody Production and CD4+ T Cell-Related Gene Responses in the Autoimmune NZBWF1 Mouse

Mortality from systemic lupus erythematosus (SLE), a prototypical autoimmune disease, correlates with the onset and severity of kidney glomerulonephritis. There are both preclinical and clinical evidence that SLE patients may benefit from consumption of n-3 polyunsaturated fatty acids (PUFA) found in fish oil, but the mechanisms remain unclear. Here we employed the NZBWF1 SLE mouse model to compare the effects of dietary lipids on the onset and severity of autoimmune glomerulonephritis after consuming: 1) n-3 PUFA-rich diet containing docosahexaenoic acid-enriched fish oil (DFO), 2) n-6 PUFA-rich Western-type diet containing corn oil (CRN) or 3) n-9 monounsaturated fatty acid (MUFA)-rich Mediterranean-type diet containing high oleic safflower oil (HOS). Elevated plasma autoantibodies, proteinuria and glomerulonephritis were evident in mice fed either the n-6 PUFA or n-9 MUFA diets, however, all three endpoints were markedly attenuated in mice that consumed the n-3 PUFA diet until 34 wk of age. A focused PCR array was used to relate these findings to the expression of 84 genes associated with CD4+ T cell function in the spleen and kidney both prior to and after the onset of the autoimmune nephritis. n-3 PUFA suppression of autoimmunity in NZBWF1 mice was found to co-occur with a generalized downregulation of CD4+ T cell-related genes in kidney and/or spleen at wk 34. These genes were associated with the inflammatory response, antigen presentation, T cell activation, B cell activation/differentiation and leukocyte recruitment. Quantitative RT-PCR of representative affected genes confirmed that n-3 PUFA consumption was associated with reduced expression of CD80, CTLA-4, IL-10, IL-18, CCL-5, CXCR3, IL-6, TNF-α and osteopontin mRNAs in kidney and/or spleens as compared to mice fed n-6 PUFA or n-9 MUFA diets. Remarkably, many of the genes identified in this study are currently under consideration as biomarkers and/or biotherapeutic targets for SLE and other autoimmune diseases.

Extensive Lesions of Monkeypox in a Prairie Dog (Cynomys sp.)

Monkeypox with extensive lesions was diagnosed in a prairie dog that was involved in a recent human outbreak of monkeypox in the Midwestern United States. Gross lesions included oral ulcers, pulmonary consolidation, enlarged cervical and thoracic lymph nodes, and multifocal, small, white umbilicated plaques in the gastrointestinal wall. Microscopic lesions were extensive in the lungs and consisted of fibrinonecrotic bronchopneumonia with vasculitis and poorly defined eosinophilic intracytoplasmic inclusion bodies in cells thought to be alveolar epithelial cells, histiocytes, and fibroblasts. Multifocal necrotizing lesions, often accompanied by myxedema, were also present in most of the other examined organs. Aggregates of pox viral particles were observed within lesions by transmission electron microscopy. Monkeypox virus infection was confirmed by real-time polymerase chain reaction and virus culture at the Centers for Disease Control and Prevention. This report highlights the difficulties of rapid diagnosis of exotic or emerging diseases and further substantiates the prairie dog as an animal model of monkeypox.