PET/MR Imaging in Evaluating Treatment Failure of Head and Neck Malignancies: A Neck Imaging Reporting and Data System-Based Study.

BACKGROUND AND PURPOSE: PET/MR imaging is a relatively new hybrid technology that holds great promise for the evaluation of head and neck cancer. The aim of this study was to assess the performance of simultaneous PET/MR imaging versus MR imaging in the evaluation of posttreatment head and neck malignancies, as determined by its ability to predict locoregional recurrence or progression after imaging. MATERIALS AND METHODS: The electronic medical records of patients who had posttreatment PET/MR imaging studies were reviewed, and after applying the exclusion criteria, we retrospectively included 46 studies. PET/MR imaging studies were independently reviewed by 2 neuroradiologists, who recorded scores based on the Neck Imaging Reporting and Data System (using CT/PET-CT criteria) for the diagnostic MR imaging sequences alone and the combined PET/MR imaging. Treatment failure was determined with either biopsy pathology or initiation of new treatment. Statistical analyses including univariate association, interobserver agreement, and receiver operating characteristic analysis were performed. RESULTS: There was substantial interreader agreement among PET/MR imaging scores (κ = 0.634; 95% CI, 0.605-0.663). PET/MR imaging scores showed a strong association with treatment failure by univariate association analysis, with P < .001 for the primary site, neck lymph nodes, and combined sites. Receiver operating characteristic curves of PET/MR imaging scores versus treatment failure indicated statistically significant diagnostic accuracy (area under curve range, 0.864-0.987; P < .001). CONCLUSIONS: Simultaneous PET/MR imaging has excellent discriminatory performance for treatment outcomes of head and neck malignancy when the Neck Imaging Reporting and Data System is applied. PET/MR imaging could play an important role in surveillance imaging for head and neck cancer.

Successful development of methodology for detection of hapten-specific contact hypersensitivity (CHS) memory in swine.

Hapten contact hypersensitivity (CHS) elicits a well-documented inflammation response that can be used to illustrate training of immune cells through hapten-specific CHS memory. The education of hapten-specific memory T cells has been well-established, recent research in mice has expanded the "adaptive" characteristic of a memory response from solely a function of the adaptive immune system, to innate cells as well. To test whether similar responses are seen in a non-rodent model, we used hapten-specific CHS to measure the ear inflammation response of outbred pigs to dinitrofluorobenzene (DNFB), oxazolone (OXA), or vehicle controls. We adapted mouse innate memory literature protocols to the domestic pig model. Animals were challenged up to 32 days post initial sensitization exposure to the hapten, and specific ear swelling responses to this challenge were significant for 7, 21, and 32 days post-sensitization. We established hapten-specific CHS memory exists in a non-rodent model. We also developed a successful protocol for demonstrating these CHS responses in a porcine system.

Evaluating the cytokine profile of the WC1+ γδ T cell subset in the ileum of cattle with the subclinical and clinical forms of MAP infection.

In the present study, we evaluated expression of IFN-γ, IL-17, TNF-α, IL-10 and TGF-ß by mucosal cells, including WC1+ γδ T cells, in ileal tissues taken from non-infected cattle and cattle naturally infected with Mycobacterium avium subsp paratuberculosis (MAP). Infected cattle were either in the subclinical or clinical stage of infection. We hypothesized that the cytokine profile of the WC1+ γδ T cell subset would be different between subclinical and clinical cattle. Our data indicate a significant increase in the numbers of WC1+ γδ T cells expressing IL-10 in clinical cattle compared to subclinical and non-infected cattle. We observed a significant increase in TGF-ß expression by non-WC1+ cells in clinically infected cattle. Expression of IFN-γ, IL-17 and TNF-α in mucosal cells, including the WC1+ γδ T cell subset, was identified in all examined groups. However, our data indicate that the stage of infection did not significantly influence expression of these proinflammatory cytokines. This study demonstrates changes in the cytokine mRNA expression profile of mucosal cells in the ileum, and specifically WC1+ γδ T cells, as cattle progress to the clinical disease. The change is characterized by an increase in expression of anti-inflammatory cytokines.

WC1+ γδ T cells from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis respond differentially to stimulation with PPD-J.

A role for γδ T cells in protection against mycobacterial infections including Johne's disease (JD) has been suggested. In neonatal calves where the risk to infection with Mycobacterium avium subsp. paratuberculosis (MAP) is high, the majority of circulating CD3+ lymphocytes are γδ TCR+. Bovine γδ T cells are divided into two major subsets based on the surface expression of workshop cluster 1 (WC1). The WC1+ subset, the predominant subset in periphery, is further divided into WC1.1+ and WC1.2+ subpopulations. The ability of γδ T cells to produce IFN-γ prior to CD4+ αß T cell activation could be crucial to the outcome of MAP infection. In the current study, cattle were naturally infected with MAP and were classified as either in the subclinical or clinical stage of infection. Compared to the control non-infected group, γδ T cell frequency in circulating lymphocytes was significantly lower in the clinical group. The observed decline in frequency was restricted to the WC1.2+ subset, and was not associated with preferential migration to infection sites (distal-ileum). γδ T cells proliferated significantly in recall responses to stimulation with purified protein derivative from MAP (PPD-J) only in subclinically infected cattle. These responses were a heterogeneous mixture of WC1.1 and WC1.2 subsets. Proliferation and IFN-γ production by the WC1.1+ γδ T cell subset was significantly higher in the subclinical group compared to the control and clinical groups. Our data indicates differences in MAP-specific ex-vivo responses of peripheral WC1+ γδ T cells of cattle with the subclinical or clinical form of JD.

Bcl-2/caspase 3 mucosal imbalance favors T cell resistance to apoptosis in dogs with inflammatory bowel disease.

Canine idiopathic inflammatory bowel disease (IBD) is believed to result from complex interplay between genetic, microbial, and immunologic factors. Abnormal cell death by apoptosis may result in the persistence of activated intestinal T cells that contribute to mucosal inflammation and clinical severity. To test this hypothesis, we investigated the mucosal expression of pro- and anti-apoptotic proteins in different intestinal compartments and their association with inflammatory indices in dogs with IBD. Apoptosis of lamina propria (LP) T cells in duodenal, ileal, and colonic tissues in control and IBD dogs was analyzed by caspase 3/Bcl-2 immunohistochemistry and TUNEL assays. Densities and distributions of LP caspase 3 and Bcl-2 cells were correlated to histopathologic lesions and the clinical activity index (CIBDAI). Compared to control tissues, IBD dogs had significantly (P<0.01) fewer caspase 3 cells in colonic mucosa. Double immunostaining identified the majority of apoptotic cells as TUNEL(+)/caspase 3(+). Within intestinal mucosa of IBD dogs, there were significantly greater numbers of Bcl-2 cells at the apical and basilar villus in the duodenum as compared to the colon and to the apical and basilar villus in the ileum (P<0.001 for all comparisons). There were significantly greater numbers of Bcl-2 cells at the apical and basilar villus of the duodenum but significantly fewer numbers of Bcl-2 cells at the apical villus of the ileum in IBD dogs compared with controls (P<0.001, P<0.001, and P<0.02, respectively). There was a significant association between the number of Bcl-2 cells in the duodenum of IBD dogs and the CIBDAI (P<0.001 each for mild, moderate and severe clinical IBD). In conclusion, apoptosis of T lymphocytes varies within intestinal compartments of dogs with IBD. Mucosal imbalance of Bcl-2/caspase 3 expression favors T cell resistance to apoptosis which may contribute to T cell accumulation and chronic intestinal inflammation, similar to human IBD.

Design of a simplified histopathologic model for gastrointestinal inflammation in dogs.

Significant interobserver variability in the diagnostic interpretation of endoscopic gastrointestinal (GI) specimens exists even with the use of World Small Animal Veterinary Association (WSAVA) standardization criteria. Chi-square analyses compared the extent of pathologists' agreement for microarchitectural features of inflammation in endoscopic specimens obtained from 253 animals of the original WSAVA study. Patterns of agreement between pathologists were classified as broad (3/4 pathologists agreed), dichotomous (2/4 pathologists agreed), or divergent (no agreement between pathologists). The simplified model for GI inflammation was based on those parameters for which the pathologists had either broad or minimally divergent opinions of histopathologic significance. In this model, the parameters chosen were as follows: gastric parameters (intraepithelial lymphocytes [IELs], lamina propria [LP] infiltrates, and mucosal fibrosis), duodenal parameters (villus atrophy, epithelial injury, IELs, crypt changes, and LP infiltrates), and colonic parameters (epithelial injury, crypt dilation, fibrosis, LP infiltrates, and goblet cell depletion). Preliminary data using this simplified model showed excellent correlation between pathologists in defining the presence and extent of GI inflammation in dogs.

Gamma-delta T-cell responses during subcutaneous Mycobacterium avium subspecies paratuberculosis challenge in sensitized or naive calves using matrix biopolymers.

We have developed a model to explore the early immune response against Mycobacterium avium subspecies paratuberculosis (Map) infection in the bovine calf using subcutaneously placed liquid gel matrix biopolymer (matrigel) containing live Map. Matrigel rapidly polymerizes in vivo, retains recruited cellular infiltrates and soluble immune mediators, and can be rapidly removed 48 hours later and depolymerized for analysis. In this study, we examined early host immune events at matrigel/Map sites; recruited cells were evaluated by histopathology and flow cytometry, and cytokines were measured by flow cytometry, enzyme-linked immunosorbent assay, and Luminex bead immunoassay. Our results demonstrate earlier recruitment of gamma-delta (γδ) T cells to matrigel/Map challenge sites compared to CD4+ T cells. We also show that significantly more γδ T cells were recruited to matrigel/Map sites postinfection day 7 compared to postinfection day 30 and that these cells produced significant amounts of the cytokine interferon gamma. We also provide evidence that peripheral blood-derived γδ T-cell subsets in cattle differentially generate interferon gamma, suggesting distinct roles for these cells. These data provide unique insight into initial antimycobacterial host cellular immune responses following Map infection in calves.

Safety evaluation of lasalocid use in Chinese ring-necked pheasants (Phasianus colchicus).

Coccidiosis remains a significant threat to the welfare of game farm-reared pheasants in the United States. Although lasalocid has been demonstrated to be effective against pheasant specific coccidia, information regarding its safety in this species is lacking. The purpose of this study was to gather data on the safety of lasalocid when fed to Chinese ring-necked pheasants at one, two, and three times the recommended high dose of lasalocid used for prevention of coccidiosis in other poultry at three times the normal treatment period. Pheasant chicks (approximately 1 day-old; n = 160) were randomly blocked by sex into four treatment groups and given their respective diets continuously for 6 wk. No significant differences were observed in overall feed consumption, weight gain, feed conversion rates, clinical pathology measurements, or tissue gross and histopathologic evaluations between controls and treatment groups associated with lasalocid administration. Based on the results of this study it appears that lasalocid fed at the recommended rate of 125 ppm is safe in Chinese ring-necked pheasants.

Direct inoculation of Mycobacterium avium subspecies Paratuberculosis into ileocecal Peyer's patches results in colonization of the intestine in a calf model.

The objective of this study was to develop an intestinal model of Mycobacterium avium subspecies paratuberculosis (Map) infection in the calf for evaluation of mucosal pathology and local and systemic immunologic responses. Map was inoculated into Peyer's patches of young calves using a right flank surgical approach in standing calves to exteriorize the ileocecal junction. Inoculum doses ranging from 10(3) to 10(9) colony-forming units of strain K10 Map were injected through the serosal surface into Peyer's patches of the distal ileum near the ileocecal valve. Fecal samples were collected for culture from each calf weekly until termination of the study. Calves were necropsied at 7, 30, 60, and 90 days after infection, when inoculation sites, lymph nodes, spleen, and peripheral blood were collected for evaluation. Ileocecal lymph nodes were consistently colonized by Map in the 10(5) to 10(9) groups. The ileocecal valve was also colonized in 10(7) and 10(9) groups. This correlated with fecal culture results as infected calves intermittently shed Map in their feces throughout the study. Granulomatous lesions with giant cells and acid-fast bacilli at the ileocecal junction, ileocecal lymph nodes, and lamina propria of high-dose animals (10(7) and 10(9)) were identified from each time point. Flow cytometry was used to detect antigen-specific production of interferon-γ and interleukin-4 locally (ileocecal lymph node) and systemically (peripheral blood mononuclear cells), which defined distinct immunologic profiles in low-dose and high-dose calves. This study demonstrates intestinal Map infection via Peyer's patch inoculation, a novel model with many shared features of natural Map infection.

Pathogenesis of Mycobacterium avium subsp. paratuberculosis in neonatal calves after oral or intraperitoneal experimental infection.

Understanding the host response to Mycobacterium avium subsp. paratuberculosis is critical to the development of effective vaccines and therapeutics for the control of this disease in the field. The current study compared the effectiveness of oral and intraperitoneal (IP) methods of experimental inoculation and two strains of M. avium subsp. paratuberculosis (strain K-10 and clinical isolate 509) on the level of infection and lesion development. Calves were inoculated with 4x10(11) to 8x10(12)cfu live bacteria, depending upon treatment group. Fecal shedding of M. avium subsp. paratuberculosis was minimal and infrequent over the course of the study for calves that received strain K-10 (oral and IP), however, calves orally inoculated with the clinical isolate shed high numbers of bacteria in their feces up to 4 months post-inoculation. Colonization was present in a number of intestinal tissues and lymph nodes with the lowest number of affected tissues in the IP calves and the highest for calves receiving the clinical isolate via oral inoculation. Microscopic lesions were predominantly found in the ileal and jejunal sections of small intestine and their associated lymph nodes, as well as the ileocecal valve and node. These data suggest that a variety of experimental infection regimes can be effective but oral inoculation with a clinical isolate may result in greater colonization of tissues and fecal shedding of M. avium subsp. paratuberculosis.

Regulation of surfactant protein and defensin mRNA expression in cultured ovine type II pneumocytes by all-trans retinoic acid and VEGF.

Beta-defensins and surfactant proteins are components of the pulmonary innate immune system. Their gene expression is regulated by development, hormones, growth and immunoregulatory factors. It was our hypothesis that growth and differentiation factors such as all-trans retinoic acid (RA) and vascular endothelial growth factor (VEGF) may affect expression of selected innate immune genes by respiratory epithelial cells. Ovine JS7 cells (alveolar type II pneumocytes) were incubated in serum-free Dulbecco's modified Eagle's medium (DMEM) complete media that contained: no treatment (negative control), RA (500 nM), or VEGF (100 ng/ml) for 6, 12 or 24 h incubation. Total RNA was isolated, cDNA synthesized, and relative mRNA levels of surfactant protein A (SP-A) and SP-D, and sheep beta-defensin-1 (SBD-1) were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Cells had significantly increased expression of SP-D mRNA at 6 h and 24 h, decreased expression of SP-A mRNA at 12 h, and unchanged levels of SBD-1 mRNA after the treatment with RA compared with their respective negative controls. VEGF did not alter the expression of the three innate immune genes. These findings suggest that SP-A and SP-D have different transcription regulation pathways, and that expression of SBD-1 is not inducible by RA similar to its human homolog HBD-1. The lack of changes induced by VEGF treatment suggests that VEGF does not have a direct effect on epithelial cells, but may affect gene expression indirectly.

Mycobacterium avium subspecies paratuberculosis infection of cattle does not diminish peripheral blood-derived macrophage mycobactericidal activity.

Ruminants infected with Mycobacterium avium subspecies paratuberculosis consistently develop a multibacillary form of disease that is centered on the ileum. Mechanisms responsible for failure of macrophage function during multibacillary disease are incompletely characterized. Our data suggest that mycobactericidal functions are present, and potentially enhanced, in monocyte-derived macrophages from M. avium subsp. paratuberculosis infected cattle. Addition of CD4(+) T cells from infected animals to autologous in vitro infected macrophages did not increase bacterial killing. In contrast, CD4(+) T cells from non-infected animals did increase bacterial killing in autologous macrophages. In macrophages from both infected and non-infected cattle, bacterial killing appeared to be independent of interferon-gamma (IFN-gamma) and nitric oxide production.

Opsonization effects on Mycobacterium avium subsp. paratuberculosis--macrophage interactions.

High antibody titers in ruminants infected with Mycobacterium avium subsp. paratuberculosis correlates with disease progression. Effects of humoral responses during mycobacterial infection are not completely understood. This study suggests that activation status may be an important factor in determining macrophage ability to limit proliferation of opsonized M. avium subsp. paratuberculosis.

Inducible nitric oxide synthase immunoreactivity in the granulomatous intestinal lesions of naturally occurring bovine Johne's disease.

Inducible nitric oxide synthase (iNOS) is important in the control of a number of intracellular pathogens, including mycobacteria, and is a marker of classic macrophage activation. In human granulomatous diseases such as leprosy, a spectrum of granulomatous lesions is described, ranging from the tuberculoid to lepromatous types. Tuberculoid granulomas are associated with enhanced iNOS production and improved clinical outcomes over the lepromatous types. The aim of this study is to determine whether an association exists between morphology of bovine Johne's disease granulomas and lesion macrophage effector functions. To accomplish this, we retrospectively evaluated 24 cases of bovine Johne's disease. In each case, we recorded the predominant granuloma morphology and evaluated iNOS immunoreactivity and bacterial burden by acid-fast stains and mycobacterial immunolabeling. The results of this study demonstrate that all cases had granulomas with features most similar to the lepromatous type. This morphology correlated with heavy bacterial burdens demonstrated by acid-fast staining and mycobacterial immunoreactivity. None of the cases had high expression of iNOS in mycobacterial-positive granulomas. When iNOS immunoreactivity was identified, it was usually located near the crypts and was distinct from the granulomatous foci.

The expanding role of antiplatelet agents in coronary artery disease. A current review of aspirin, glycoprotein IIb/IIIa inhibitors, and the thienopyridines.

The platelet has assumed an increasingly important role in cardiovascular medicine as our understanding of the pathophysiology of acute coronary syndromes (ACS) has evolved. Plaque rupture, platelet aggregation, and thrombus formation occur as a result of complex interaction between the platelet, the endothelium, and various inflammatory cells and circulating proteins. Aspirin continues to form the foundation of any anti-ischemic regimen, but cardiologists have long recognized the need for newer, more potent antiplatelet agents. Glycoprotein IIb/IIIa receptor antagonists and thienopryidines have been developed over the past decade and now serve as powerful complements to aspirin in the prevention and treatment of coronary events. The paper will begin with a review of aspirin as well as a discussion of the concept of aspirin resistance. The rapidly expanding body of knowledge supporting the use of glycoprotein IIb/IIIa receptor blockers and thienopyridines will then be addressed, with an emphasis on reconciling recent controversies in the literature. Future advances in the treatment of coronary artery disease will likely occur as we further refine the role of these established antiplatelet drugs and develop agents that bind to novel targets in the thrombotic cascade.

Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells.

The mechanisms by which Mycobacterium avium subspecies paratuberculosis (M. a. ptb) survives within macrophages are not well characterized. One strategy for intracellular survival developed by Mycobacterium tuberculosis is inhibition of phagosomal maturation. In this study it was hypothesized that M. a. ptb is capable of survival within macrophages by residing within a phagosomal compartment that does not mature into a functional phagolysosome. To test this hypothesis the following objectives were determined. Phagosomal maturation was evaluated by comparison of stage specific markers on the membranes of phagosomes containing live M. a. ptb with those containing killed M. a. ptb, Mycobacterium smegmatis, and zymosan A using immunofluorescent labeling and confocal microscopy. Intracellular survival of live M. a. ptb within J774 macrophages was compared to that of M. smegmatis by direct determination of bacterial viability by differential live/dead staining. The results of this study show that the phagosomes containing live M. a. ptb had increased levels of an early marker (transferrin receptor [TFR]) and decreased levels of a late maturation marker (lysosome associated membrane protein one [Lamp-1]), relative to those containing killed M. a. ptb, M. smegmatis, and zymosan A. Additionally, compared to M. smegmatis, M. a. ptb has enhanced ability to survive within cultured macrophages. These data indicate that M. a. ptb resists intracellular killing by residing within a phagosomal compartment that retains the characteristics of early phagosomes and resists maturation into functional phagolysosome.

The biology of dendritic cells and their potential use in veterinary medicine.

Dendritic cells have been shown to be the main antigen-presenting cells in vitro and in vivo, playing a pivotal role in the induction of both tolerance and immunity. Dendritic cells from humans and mice have been extensively studied and dendritic cell-based vaccines have been shown to be effective in the prevention and treatment of infectious, allergic and neoplastic diseases. Studies of dendritic cells of domestic animal origin are becoming available and confirm a role for these cells in the pathogenesis of a variety of animal diseases, suggesting that dendritic cells could be used as adjuvants for prophylactic and therapeutic strategies in veterinary medicine.

Intracellular trafficking of Mycobacterium avium ss. paratuberculosis in macrophages.

The granulomatous enteric lesions of cattle with Johne's disease are composed of infected macrophages, and grow by accumulation, re-infection, and expansion of macrophage populations in the intestinal wall. We have examined the growth of bacteria in macrophages to define characteristics of intracellular trafficking for exocytosis, replication, and antigen presentation. Using immunocytochemical markers for light, confocal and electron microscopy, we have examined potential pathway tropisms using data for bacterial attachment, phagosomal acidification, phagolysosomal degradation and apoptosis. Our hypotheses are that pathogenic/wild-type strains block phagosomal acidification so that the phagosome fails to obtain markers of the late phagosome and phagolysosome, and this leads to the replication pathway within bacteriophorous vacuoles. Non-pathogenic strains appear to be processed to exocytosis, and avirulent mutant strains may be degraded and have preference of antigen processing pathways that involve transport vesicles bearing MHC II antigens. Pathogenicity in a nude mouse model of intestinal infection reveals lesion development and confirms pathway preferences of virulent strains for bacteriophorous vacuole formation.

Sex differences in dopamine receptor overproduction and elimination.

Density of dopamine D1 and D2 family receptors was assessed using autoradiography in male and female rats from 25 to 120 days of age, focusing on transitions through puberty into full adulthood. Males had greater overproduction (approximately 4.6-fold) and elimination of striatal D1 and D2 receptors than females, though their adult densities were very similar. Males had more extensive overproduction of D1 receptors in nucleus accumbens and sustained a greater density into adulthood (57.8 +/- 21.2%). These results have implications for understanding gender differences in the prevalence of clinical disorders associated with dopamine.