Cross Lineage Rearrangement in Feline Enteropathy-Associated T-cell Lymphoma.

Feline enteropathy-associated T-cell lymphoma (EATL) type II is characterized by infiltration of the small intestinal mucosa with small T-cells with variable epitheliotropism and is often difficult to differentiate from inflammation. Polymerase chain reaction (PCR) to assess antigen receptor rearrangements (PARR) amplifies the T- (T-cell receptor gamma, TCRG) or B-cell (immunoglobulin heavy chain, IGH) antigen receptor genes and is used to differentiate EATL from inflammation. However, PARR does not determine lymphocyte phenotype, and clonal rearrangement of either or both the TCRG or IGH genes may be detected in neoplastic T-cells. The purpose of this study was to determine the incidence of cross lineage rearrangement in feline EATL type II. Using a diagnostic algorithm combining histology, immunohistochemistry, and PARR testing, 8 of 92 cases diagnosed as EATL type II at Michigan State University between January 2013 and June 2014 showed cross lineage rearrangement (8.7%). PARR for the IGH gene facilitates the diagnosis of cases histologically highly suggestive of EATL type II in which polyclonal rearrangement of the TCRG gene is detected.

Canine Circovirus 1 (CaCV-1) and Canine Parvovirus 2 (CPV-2): Recurrent Dual Infections in a Papillon Breeding Colony.

Recurrent outbreaks of sudden death and bloody diarrhea were reported in March 2013 and February 2014 in a breeding colony of Papillon dogs. During the first outbreak, 1 adult dog and 2 eight-month-old puppies died. During the second outbreak, 2 ten-week-old puppies died. One puppy from the first outbreak and 2 puppies from the second outbreak were examined at necropsy. Histologically, all 3 puppies had severe segmental crypt necrosis of the small intestine and marked lymphoid follicle depletion in the spleen and Peyer's patches. Real-time (RT) polymerase chain reaction (PCR) demonstrated abundant canine parvovirus (CPV-2) DNA (Ct<15) in the affected small intestine, and immunohistochemistry detected large amounts of CPV-2 antigen in intestinal crypt epithelium and Kupffer cells but few positive macrophages in lymphoid organs. All puppies had marked sinusoidal histiocytosis and multifocal granulomatous inflammation in mesenteric lymph nodes and spleen, prompting additional RT-PCR testing for canine circovirus 1 (CaCV-1). Very high levels of CaCV-1 DNA (Ct<13) were detected in small intestine, lymph nodes, and spleen. In situ hybridization for CaCV-1 detected rare positive nuclei of regenerating crypt epithelium but abundant amounts of CaCV-1 nucleic acid in the cytoplasm and nuclei of histiocytes in all lymphoid tissues, including granulomatous inflammatory foci and hepatic Kupffer cells. Significant levels of CaCV-1 DNA were detected in blood and serum (Ct as low as 13) but not feces from 3 surviving dogs at 2 months or 1 year after the outbreak, respectively. We hypothesize that CPV-2 infection predisposed dogs to CaCV-1 infection and ultimately resulted in more severe clinical disease.

Distinguishing Intestinal Lymphoma From Inflammatory Bowel Disease in Canine Duodenal Endoscopic Biopsy Samples.

Inflammatory bowel disease (IBD) and intestinal lymphoma are intestinal disorders in dogs, both causing similar chronic digestive signs, although with a different prognosis and different treatment requirements. Differentiation between these 2 conditions is based on histopathologic evaluation of intestinal biopsies. However, an accurate diagnosis is often difficult based on histology alone, especially when only endoscopic biopsies are available to differentiate IBD from enteropathy-associated T-cell lymphoma (EATL) type 2, a small cell lymphoma. The purpose of this study was to evaluate the utility of histopathology; immunohistochemistry (IHC) for CD3, CD20, and Ki-67; and polymerase chain reaction (PCR) for antigen receptor rearrangement (T-cell clonality) in the differential diagnosis of severe IBD vs intestinal lymphoma. Endoscopic biopsies from 32 dogs with severe IBD or intestinal lymphoma were evaluated. The original diagnosis was based on microscopic examination of hematoxylin and eosin (HE)-stained sections alone followed by a second evaluation using morphology in association with IHC for CD3 and CD20 and a third evaluation using PCR for clonality. Our results show that, in contrast to feline intestinal lymphomas, 6 of 8 canine small intestinal lymphomas were EATL type 1 (large cell) lymphomas. EATL type 2 was uncommon. Regardless, in dogs, intraepithelial lymphocytes were not an important diagnostic feature to differentiate IBD from EATL as confirmed by PCR. EATL type 1 had a significantly higher Ki-67 index than did EATL type 2 or IBD cases. Based on the results of this study, a stepwise diagnostic approach using histology as the first step, followed by immunophenotyping and determining the Ki67 index and finally PCR for clonality, improves the accuracy of distinguishing intestinal lymphoma from IBD in dogs.

Beyond H&E: integration of nucleic acid-based analyses into diagnostic pathology.

Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets.

Distribution of eastern equine encephalomyelitis viral protein and nucleic acid within central nervous tissue lesions in white-tailed deer (Odocoileus virginianus).

An outbreak of eastern equine encephalomyelitis (EEE) occurred in Michigan free-ranging white-tailed deer (Odocoileus virginianus) during late summer and fall of 2005. Brain tissue from 7 deer with EEE, as confirmed by reverse transcriptase polymerase chain reaction, was studied. Detailed microscopic examination, indirect immunohistochemistry (IHC), and in situ hybridization (ISH) were used to characterize the lesions and distribution of the EEE virus within the brain. The main lesion in all 7 deer was a polioencephalomyelitis with leptomeningitis, which was more prominent within the cerebral cortex, thalamus, hypothalamus, and brainstem. In 3 deer, multifocal microhemorrhages surrounded smaller vessels with or without perivascular cuffing, although vasculitis was not observed. Neuronal necrosis, associated with perineuronal satellitosis and neutrophilic neuronophagia, was most prominent in the thalamus and the brainstem. Positive IHC labeling was mainly observed in the perikaryon, axons, and dendrites of necrotic and intact neurons and, to a much lesser degree, in glial cells, a few neutrophils in the thalamus and the brainstem, and occasionally the cerebral cortex of the 7 deer. There was minimal IHC-based labeling in the cerebellum and hippocampus. ISH labeling was exclusively observed in the cytoplasm of neurons, with a distribution similar to IHC-positive neurons. Neurons positive by IHC and ISH were most prominent in the thalamus and brainstem. The neuropathology of EEE in deer is compared with other species. Based on our findings, EEE has to be considered a differential diagnosis for neurologic disease and meningoencephalitis in white-tailed deer.

Polyomavirus-associated nephritis in 2 horses.

Polyomaviruses produce latent and asymptomatic infections in many species, but productive and lytic infections are rare. In immunocompromised humans, polyomaviruses can cause tubulointerstitial nephritis, demyelination, or meningoencephalitis in the central nervous system and interstitial pneumonia. This report describes 2 Standardbred horses with tubular necrosis and tubulointerstitial nephritis associated with productive equine polyomavirus infection that resembles BK polyomavirus nephropathy in immunocompromised humans.

Cutaneous lymphomas in European pet rabbits (Oryctolagus cuniculus).

Cutaneous lymphoma is a common skin neoplasm of pet rabbits in Europe but is rarely reported in pet rabbits in North America. These neoplasms have not been previously characterized, nor has the cause for the apparent predilection for cutaneous lymphoma in European pet rabbits compared with North American pet rabbits been investigated. In this retrospective study, the authors morphologically and immunohistochemically characterized 25 cutaneous lymphomas in European pet rabbits according to the World Health Organization classification. Tumors were classified as diffuse large B cell lymphomas, with 14 lymphomas exhibiting a centroblastic/centrocytic subtype and 11 tumors exhibiting a T cell-rich B cell subtype. To investigate a potential viral etiology of these lymphomas, 3 diffuse large B cell and 3 T cell-rich B cell lymphomas were evaluated by polymerase chain reaction for retroviral and herpesviral genes. Neither virus was detected. In contrast to other domestic animals, cutaneous lymphomas in European pet rabbits were highly pleomorphic and frequently contained multinucleated giant cells. Unexpectedly, the second most common subtype was T cell-rich B cell lymphoma, a subtype that is rare in species other than horses. Based on a limited number of samples, there was no support for a viral etiology that would explain the higher incidence of lymphoma in European pet rabbits compared with American pet rabbits. Further investigation into genetic and extrinsic factors associated with the development of these tumors is warranted.

Diagnostic algorithm to differentiate lymphoma from inflammation in feline small intestinal biopsy samples.

Differentiating between inflammatory bowel disease (IBD) and small intestinal lymphoma in cats is often difficult, especially when only endoscopic biopsy specimens are available for evaluation. However, a correct diagnosis is imperative for proper treatment and prognosis. A retrospective study was performed using surgical and endoscopic intestinal biopsy specimens from 63 cats with a history of chronic diarrhea or vomiting or weight loss. A diagnosis of lymphoma or inflammation was based on microscopic examination of hematoxylin and eosin (HE)-stained sections alone, HE-stained sections plus results of immunohistochemical labeling (IHC) for CD3e and CD79a, and HE staining, immunophenotyping, and polymerase chain reaction (PCR) results for B and/or T cell clonality. In addition, various histomorphologic parameters were evaluated for significant differences between lymphoma and IBD using Fisher's exact test. The sensitivity and specificity of each parameter in the diagnosis of lymphoma were also determined. Results of Bayesian statistical analysis demonstrated that combining histologic evaluation of small intestinal biopsy specimens with immunophenotyping and analysis of clonality of lymphoid infiltrates results in more accurate differentiation of neoplastic versus inflammatory lymphocytes. Important histologic features that differentiated intestinal lymphoma from IBD included lymphoid infiltration of the intestinal wall beyond the mucosa, epitheliotropism (especially intraepithelial nests and plaques), heterogeneity, and nuclear size of lymphocytes. Based on the results of this study, a stepwise diagnostic algorithm that first uses histologic assessment, followed by immunophenotyping and then PCR to determine clonality of the lymphocytes, was developed to more accurately differentiate between intestinal lymphoma and IBD.

Detection of group C rotavirus in juvenile ferrets (Mustela putorius furo) with diarrhea by reverse transcription polymerase chain reaction: sequencing and analysis of the complete coding region of the VP6 gene.

Nine juvenile ferrets (Mustela putorius furo) with a history of diarrhea were severely dehydrated and had distended abdomens and thin-walled small intestines that contained gas and fluid. Histologically, small intestines exhibited acute superficial atrophic enteritis. Transmission electron microscopy of the small intestine showed rotavirus-like particles within apical vacuoles. Reverse transcription polymerase chain reaction (RT-PCR) was negative for group A rotavirus. A group C rotavirus-specific RT-PCR assay was developed using consensus primers designed from the alignment of VP6 gene sequences of porcine, bovine, and human strains. A 182-bp product of the VP6 gene was sequenced and showed significant similarity to group C rotavirus VP6 sequences. This strain was designated "Ferret Rota C-MSU." The entire coding sequence of VP6 was determined and compared with other rotaviruses. Ferret Rota C-MSU virus was found to be most closely related to Shintoku group C rotavirus. This is the first definitive identification of a group C rotavirus in ferrets, based upon RT-PCR, sequencing, and genetic analysis.

Meningoencephalitis in a polar bear caused by equine herpesvirus 9 (EHV-9).

A 12-year-old female polar bear (Ursus maritimus) developed a sudden onset of muscle tremors, erratic circling, increased blinking, head shaking, and ptyalism, which progressed to partial and generalized seizures. Ancillary diagnostic tests were inconclusive, and the only significant laboratory finding was nonsuppurative pleocytosis of cerebrospinal fluid. Euthanasia was elected. Microscopic evaluation demonstrated multifocal, random nonsuppurative meningoencephalitis involving most prominently the rostral cerebral cortex, as well as the thalamus, midbrain, and rostral medulla. Lesions consisted of inflammation, neuronal necrosis, gliosis, and both neuronal and glial basophilic intranuclear inclusion bodies. Immunohistochemistry with a polyclonal antibody reactive to several equine herpesviruses was positive within affected areas of the brain, and polymerase chain reaction conclusively demonstrated the presence of only equine herpesvirus 9. The clinical and morphologic features of this case resemble other fatal herpesvirus encephalitides derived from interspecies transmission and underscore the need for extreme caution when managing wild or captive equids.

Peliod hepatocellular carcinoma in a domesticated ferret (Mustela putorius furo).

Peliod hepatocellular carcinoma was diagnosed in a domesticated ferret (Mustela putorius furo). The diagnosis was made using immunohistochemical analysis, histologic examination, and the accepted classification schemes based on histomorphologic features. Bilateral, adrenocortical hyperplasia also was evident. Speculation about a possible association between the variant of hepatocellular neoplasia diagnosed in this animal and its adrenal pathologic changes was done.

[Electronical podometry: EMD system]. / La podométrie electronique: la plate-forme EMED.

The loads supported by the feet can be appreciated by the analysis of footprints. X-rays of the feet in weight-bearing complete the static analysis of the foot. The footprints and X-rays of the feet allow to obtain a static assessment of the loads applied on the foot. With the evolution of the technology, the EMED system for measuring dynamic foot pressures is appeared. The EMED system with the different clinical applications is described.

[Study of the plantar arch: correlations between podometrical and radiological parameters. results of a prospective study of 79 cases]. / Etude de la voûte plantaire: corrélations entre des paramètres podométriques et radiologiques. Résultats d'une etude prospective de 79 cas.

The authors present a prospective study on the potential correlations between eight footprint parameters and three radiological parameters in the study of the plantar arch. Seventy nine patients were evaluated in 2001. The eight footprint parameters were as fellows : the arch angle, the Chippaux-Smirak's index, the Quamra's contact index 2,3 and 4, the Schwartz's footprint angle, the Staheli's arch index and the arch length index. The three radiological parameters were as fellows : the Djian-Annonier's angle, the Méary's angle and the calcaneal inclination. This prospective study confirms the best correlation, found in others studies, obtained between the Djian-Annonier's angle and the Chippaux-Smirak. The use of the Méary's angle and the calcaneal inclination is not justified because they have bad correlations with footprint parameters.

Phase I clinical and pharmacokinetic study of topotecan administered by a 24-hour continuous infusion.

PURPOSE: To determine the maximum-tolerable dose (MTD) and to investigate the pharmacokinetics and pharmacodynamics of topotecan in a phase I study. Topotecan is a novel semisynthetic derivative of the anticancer agent camptothecin and inhibits the intranuclear enzyme topoisomerase I. Broad preclinical activity rationalized further clinical evaluation. PATIENTS AND METHODS: In this phase I trial, topotecan was administered by 24-hour continuous infusion every 21 days to patients with solid malignant tumors. RESULTS: A total of 25 eligible patients, of whom 22 were pretreated, entered the study. They received the following dosages of topotecan: 2.5, 3.75, 5.60, 8.4, and 10.5 mg/m2 by 24-hour infusion. Reversible leukopenia and thrombocytopenia were dose-limiting, with mild anemia occurring regularly. Other toxicities, such as alopecia, mucositis, nausea, and vomiting were sporadic and mild. Responses were not observed. However, eight patients had stable disease. The plasma concentration-time curves were not compatible with standard linear pharmacokinetic models, and indications were found for the occurrence of nonlinear (saturation) kinetics at the dosages studied. CONCLUSION: The recommended dose for phase II studies is 8.4 mg/m2 when administered as a 24-hour infusion, which is well tolerated. Further studies will be necessary to account for the putative nonlinear behavior of the drug.

Absence of 5-HT(1B) receptors is associated with impaired impulse control in male 5-HT(1B) knockout mice.

BACKGROUND: Serotonin (5-HT) plays a complex regulatory role in processes like anxiety, depression, aggression, and impulse control. Due to the large amount of serotonergic receptors, knockout mice offer an important opportunity to investigate the role of specific receptors. The 5-HT(1B) receptor is thought to mediate aggression and impulse control. This was studied here in mice lacking 5-HT(1B) receptors (5-HT(1B) KO). METHODS: Wild type and 5-HT(1B) KO mice were exposed to several types of entrained and nonentrained stimuli. With telemetry, body temperature, heart rate, and locomotor activity were measured continuously during the different experiments. RESULTS: To nonentrained stimuli like disturbance stress and confrontation with an intruder, 5-HT(1B) KO mice showed exaggerated physiologic and behavioral responses. These mice displayed behavioral disinhibition, measured as increased social interest and aggression to an intruder mouse. However, in response to well-entrained stimuli like daily light transitions, responses were smaller in 5-HT(1B) KO than in wild type mice, suggesting that hyperreactivity is stimulus specific. CONCLUSIONS: Serotonin 1B receptors are essential in impulse control by inhibiting responses to nonentrained stimuli. Therefore, the 5-HT(1B) KO mouse might be an important additional model for studying aspects of disinhibition in aggression and impulse control.

Stress-induced hyperthermia in the 5-HT(1A) receptor knockout mouse is normal.

BACKGROUND: Several studies on serotonin 1A (5-HT(1A)) receptor knockout mice in different genetic backgrounds indicate that such mice display a more anxious phenotype than their corresponding wild types. We hypothesized that the 5-HT(1A) receptor knockout mice would show a different phenotype than the wild type mice in the stress-induced hyperthermia (SIH) paradigm, which tests putative anxiolytic effects of drugs. Moreover, on pharmacologic challenges with the 5-HT(1A) receptor agonist flesinoxan we expected an absence of the functional response in knockout mice relative to wild type mice. METHODS: Effects of the 5-HT(1A) receptor agonist flesinoxan, alone or in combination with the 5-HT(1A) receptor antagonist WAY-100635, and the gamma-aminobutyric acid A (GABA(A))-benzodiazepine receptor agonist diazepam were studied in the SIH paradigm in male 129/Sv 5-HT(1A) receptor knockout and wild type mice. In addition, the effects of flesinoxan on plasma corticosterone concentrations were determined. RESULTS: Plasma corticosterone concentrations were dose dependently elevated by flesinoxan in wild type mice but not in knockout mice. Flesinoxan dose dependently decreased SIH in wild type mice but not in knockout mice. The flesinoxan effect in wild type mice was blocked by WAY-100635. Furthermore, diazepam decreased SIH in both genotypes. There were no differences in basic SIH responses between wild type and knockout mice. CONCLUSIONS: 5 -HT(1A) receptor knockout mice display a normal SIH response, and results indicate, based on the SIH, that the GABA(A)-benzodiazepine receptor complex functions normally.

Erythrocytes and the transfer of anticancer drugs and metabolites: a possible relationship with therapeutic outcome.

Blood functions as a mobile tissue in an exchange system, with the remaining body tissue as a stationary phase. The equilibrium among plasma water, plasma proteins, and blood cells is described by models, but little consideration has been given to the substance-binding capacity of erythrocytes. There are numerous reasons for this, including bioanalytical limitations (ie, it has been difficult to study erythrocytes in the laboratory in their natural state). Erythrocyte monitoring requires accurate blood sampling and quantitative isolation of erythrocytes without disturbing the equilibrium of substances of interest between erythrocytes and plasma or other blood constituents. This became possible with the advent of the measurement of sediment device. The mass of a given substance available in blood can be described by M(Blood) = M(Plasma) + M(ERY) (+ M(REM)). M(ERY) is the mass of a substance present in erythrocytes and it is shown that for several oxazaphosphorines, such as iphosphoramide mustard, that M(ERY) determines M(Blood) with great superiority over M(Plasma). The impact of erythrocyte monitoring on therapeutic outcome has to be defined, but is an important area of research.

Detection of human IgG antibodies against free N-acetyl-L-cysteine.

Human antibodies of the IgG type reacting with free N-acetyl-cysteine are detectable with an appropriate immunoconjugate. Free cysteine, homocysteine and glutathione, but not cystine, interfere with the antibodies. The interference with cysteine and homocysteine is frequently reduced after nitrosylation of these amino acids. The interference with glutathione is frequently amplified after nitrosylation of this oligopeptide. The antibodies react weakly with whole red blood cells. Autoantibodies of this type may explain the wasting observed in untreated tuberculosis patients and in AIDS patients.

Intravesical drug delivery. Pharmacokinetic and clinical considerations.

Intravesical drug administration is widely used in the treatment of patients with superficial bladder cancer, and aims to optimise drug delivery in the vicinity of the tumour and reduce systemic availability. The most commonly employed intravesical agents in patients with superficial bladder cancer are mitomycin (mitomycin C), thiotepa, ethoglucid (ethoglucid), anthracyclines such as doxorubicin, bacille Calmette-Guérin (BCG) and, more recently, taxol and the new mitomycin derivative KW-2149. Recurrence rates in patients with superficial bladder cancer have been substantially reduced by combined transurethral resection and intravesical pharmacotherapy. The high concentration of cytotoxics in urine and tumour tissue explain the high efficacy rates. Furthermore, the low systemic availability of most intravesical agents is consistent with the low frequency of acute and delayed systemic adverse effects. Systemic toxicity is almost negligible, except in the case of thiotepa, and local toxicity is transient and tolerable. Pharmacokinetic models of drug absorption from the bladder have been developed, both in animals and humans. These have led to the identification of optimal intravesical treatment regimens.

Correlation of the response to cisplatin of human ovarian cancer cell lines, originating from one tumor but with different sensitivity, with the recovery of DNA adducts.

Cis-Diamminedichloroplatinum(II) (CDDP) is used in the treatment of various cancers, with or without ionizing radiation. During treatment, resistance may develop, and cross-resistance can also occur. DNA is the main target for CDDP and ionizing radiation, and we therefore evaluated the correlation between the amount of CDDP-DNA adducts and the cytotoxic activity of CDDP in human ovarian cancer cell lines with different platinum sensitivities. DNA-adduct levels were investigated 18 hr after CDDP exposure in three cell lines originating from the same human ovarian cancer. The least sensitive cells appeared to have the largest amounts of CDDP-DNA adducts, while the most sensitive had higher adduct levels than the parental cells. The proportion of the four adducts measured (i.e., Pt-G, Pt-AG, Pt-GG, and G-Pt-G) was comparable in all cell lines, with a preference for Pt-GG adduct formation (> 50% of the adducts). Intracellular CDDP concentrations were higher in sensitive than in resistant cells, in contrast to the degree of CDDP adduct formation. Data obtained following continuous exposure of CDDP-resistant cells to CDDP suggest that DNA repair is partly responsible for resistance to CDDP. We conclude that the amount of CDDP-DNA adduct formation in cancer cells is not a predictor of CDDP cytotoxicity.