Dorothee Bienzle

Classification of Canine Malignant Lymphomas According to the World Health Organization Criteria

A study was carried out to test the accuracy and consistency of veterinary pathologists, not specialists in hematopathology, in applying the World Health Organization (WHO) system of classification of canine lymphomas. This study represents an initiative of the ACVP Oncology Committee, and the classification has been endorsed by the World Small Animal Veterinary Association (WASVA). Tissue biopsies from cases of canine lymphoma were received from veterinary oncologists, and a study by pathologists given only signalment was carried out on 300 cases. Twenty pathologists reviewed these 300 cases with each required to choose a diagnosis from a list of 43 B and T cell lymphomas. Three of the 20 were hematopathologists who determined the consensus diagnosis for each case. The 17 who formed the test group were experienced but not specialists in hematopathology, and most were diplomates of the American or European Colleges of Veterinary Pathology. The overall accuracy of the 17 pathologists on the 300 cases was 83%. When the analysis was limited to the 6 most common diagnoses, containing 80% of all cases, accuracy rose to 87%. In a test of reproducibility enabled by reintroducing 5% of cases entered under a different identity, the overall agreement between the first and second diagnosis ranged from 40 to 87%. The statistical review included 43,000 data points for each of the 20 pathologists.

Molecular Evidence Supporting Ehrlichia canis‐Like Infection in Cats

Currently, the pathogenic role of Ehrlichia canis in cats has been proposed predominantly on the basis of the serologic evidence of natural infection and the infrequent detection of morulae‐like structures within the cytoplasm of leukocytes in cats. The purpose of this report was to provide molecular evidence supporting E cams‐like infection in 3 cats that had clinical manifestations consistent with canine ehrlichiosis but lacked antibodies to E canis antigens. Serum from all 3 cats contained antinuclear antibodies (ANAs). The predominant disease manifestation was polyarthritis in 1 cat and bone marrow hypoplasia or dysplasia, accompanied by pancytopenia or anemia and thrombocytopenia, in 1 cat each. The alignment of E canis partial 16S ribosomal DNA (rDNA; 382 nucleotide positions), amplified from EDTA blood samples from each cat, was identical to each other and was identical to a canine isolate of E canis (GenBank accession number AF373613). In 1 cat, concurrent treatment with corticosteroids may have interfered with the therapeutic effectiveness of doxycycline for the elimination of E canis‐like infection. To further define the spectrum of ehrlichiosis in cats, polymerase chain reaction (PCR) testing may be necessary until serologic testing is thoroughly validated in experimentally or naturally infected cats. In addition, until E canis has been isolated from cats and several tissue culture isolates are available from disparate geographic regions for detailed comparative genetic study, the molecular evidence presented in this study supporting E canis‐like infection in cats must be interpreted with caution.

Peptidylarginine deiminase 2 (PAD2) overexpression in transgenic mice leads to myelin loss in the central nervous system

SUMMARY Demyelination in the central nervous system is the hallmark feature in multiple sclerosis (MS). The mechanism resulting in destabilization of myelin is a complex multi-faceted process, part of which involves deimination of myelin basic protein (MBP). Deimination, the conversion of protein-bound arginine to citrulline, is mediated by the peptidylarginine deiminase (PAD) family of enzymes, of which the PAD2 and PAD4 isoforms are present in myelin. To test the hypothesis that PAD contributes to destabilization of myelin in MS, we developed a transgenic mouse line (PD2) containing multiple copies of the cDNA encoding PAD2, under the control of the MBP promoter. Using previously established criteria, clinical signs were more severe in PD2 mice than in their normal littermates. The increase in PAD2 expression and activity in white matter was demonstrated by immunohistochemistry, reverse transcriptase-PCR, enzyme activity assays, and increased deimination of MBP. Light and electron microscopy revealed more severe focal demyelination and thinner myelin in the PD2 homozygous mice compared with heterozygous PD2 mice. Quantitation of the disease-associated molecules GFAP and CD68, as measured by immunoslot blots, were indicative of astrocytosis and macrophage activation. Concurrently, elevated levels of the pro-inflammatory cytokine TNF-α and nuclear histone deimination support initiation of demyelination by increased PAD activity. These data support the hypothesis that elevated PAD levels in white matter represents an early change that precedes demyelination.

Clinical, Laboratory, and Histopathologic Features of Equine Lymphoma

Clinical, laboratory and tissue findings from 37 horses with lymphoma were investigated. Horses ranged in age from 0.3 to 20.5 years (median 5.0 years) and included 18 females and 19 males. Weight loss (n = 25) and ventral edema (n = 21) were the most common historical and physical abnormalities. The most common laboratory abnormalities were hyperfibrinogenemia (n = 26), hypoalbuminemia (n = 19), anemia (n = 19), leukemia (n = 14), hyperglobulinemia (n = 13), and thrombocytopenia (n = 13). Thirty-four tumors involved multiple lymphoid tissues and abdominal or thoracic organs, and 3 tumors were restricted to cutaneous and subcutaneous sites. Histopathologically, all tumors diffusely effaced normal lymph node architecture. Tumor cell morphology was heterogeneous in 17 tumors, and 8 tumors had marked histiocytic and multinucleated giant cell infiltrates. Extensive necrosis or focal fibrosis was present in 22 and 4 lymphomas, respectively. Staining of tumor sections with antibodies against CD3 and CD79α molecules resulted in classification of T-cell (n = 26) or B-cell (n = 7) origin. Four tumors could not be classified. Most T-cell tumors comprised small to medium CD3+ lymphocytes, whereas 5 of 7 B-cell tumors were infiltrated by numerous small T lymphocytes and classified as T-cell-rich B-cell lymphoma. Neither estrogen nor progesterone receptor expression was consistently identified by immunochemical assessment of tumor tissues. Fresh tumor cells from 6 horses bound antibodies reactive with equine CD4, CD5, CD8, CD21, or major histocompatibility class II molecules, confirming T-cell (n = 5) or B-cell origin (n = 1). These findings suggest that T-cell lymphoma is more common than B-cell lymphoma in horses and that inflammation, possibly from tumor cytokine production, is frequent.

Developmental partitioning of myelin basic protein into membrane microdomains

Specific membrane microdomains (including lipid rafts) exist in myelin but have not been fully characterized. Myelin basic protein (MBP) maintains the compactness of the myelin sheath and is highly posttranslationally modified. Thus, it has been suggested that MBP might also have other functions, e.g., in signal transduction. Here, the distribution of MBP and its modified forms was studied, spatially and temporally, by detailed characterization of membrane microdomains from developing and mature bovine myelin. Myelin membranes were extracted with three different detergents (Brij 96V, CHAPS, or Triton X‐100) at 4°C. The detergent‐resistant membranes (DRMs), representing coalesced lipid rafts, were isolated as low‐buoyant‐density fractions on a sucrose density gradient. These myelin rafts were disrupted when cholesterol was depleted with methyl‐β‐cyclodextrin. The use of CHAPS detergent led to enrichment of several myelin proteins, including phospho‐Thr97‐MBP, in the DRMs from mature myelin. Citrullinated and methylated MBP remained in “nonraft” microdomains. In contrast, the DRMs from early myelin were enriched in Golli‐MBP, Fyn, Lyn, and CNP. The localization of various proteins in DRMs was further supported by the colocalization of these lipid raft components in cultured mouse oligodendrocytes. Thus, there is a developmental regulation of posttranslationally modified forms of MBP into specific membrane microdomains.

Factors Contributing to the Lack of Human Immunodeficiency Virus Type 1 (HIV-1) Transmission in HIV-1-Discordant Partners

Correlates of resistance to infection by human immunodeficiency virus type 1 (HIV-1) are important for defining potential therapeutic interventions and for prophylactic vaccination. In this study, 11 couples discordant in their HIV-1 infection status were prospectively evaluated for the presence of protective factors. Behavioral characteristics of all subjects entailed a high risk of transmission. Cytotoxic T lymphocyte (CTL) responses against viruses isolated from the infected partner, and against laboratory virus isolates, were detected in 5 (45%) of 11 HIV-negative partners, including a CCR5Delta32-homozygous and a heterozygous subject. No CTL responses were observed in 6 control unexposed subjects. Marked variation in lymphocyte susceptibility to viral infection was noted. Resistance attributable to major histocompatibility complex discordance or anti-major histocompatibility complex antibodies was not identified. These results suggest that a combination of factors, including cellular immunity, viral characteristics, and coreceptor integrity, may be involved in the persistent nontransmission of HIV.

Expressed sequence tags from Madagascar periwinkle (Catharanthus roseus)

The Madagascar periwinkle (Catharanthus roseus) is well known to produce the chemotherapeutic anticancer agents, vinblastine and vincristine. In spite of its importance, no expressed sequence tag (EST) analysis of this plant has been reported. Two cDNA libraries were generated from RNA isolated from the base part of young leaves and from root tips to select 9824 random clones for unidirectional sequencing, to yield 3327 related sequences and 1696 singletons by cluster analysis. Putative functions of 3663 clones were assigned, from 5023 non‐redundant ESTs to establish a resource for transcriptome analysis and gene discovery in this medicinal plant.

Recommended Guidelines for Submission, Trimming, Margin Evaluation, and Reporting of Tumor Biopsy Specimens in Veterinary Surgical Pathology

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

Immunophenotype and functional properties of feline dendritic cells derived from blood and bone marrow.

Dendritic cells (DCs) are a heterogeneous population of cells of fundamental importance in initiating innate as well as specific immune responses. The identity and function of DCs in the cat are unknown, although they are likely pivotal in the response to infection. In this study, feline DCs were derived by 3-10-day culture of adherent blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) in the presence of IL 4 and GM-CSF. BMMC consistently yielded a greater number of DCs than PBMC, and there were fewer macrophages than DC from both compartments. DCs expressed a distinct constellation of surface molecules, which included CD1a, CD1b, and CD1c, CD11b, CD14, and 2-3-fold higher levels of MHC class I and II molecules than co-cultured macrophages or fresh blood monocytes. DCs displayed typical cytoplasmic processes, limited non-specific esterase activity, and acquired antigen by phagocytosis, pinocytosis, and binding to specific receptors. Cytokine-exposed cells induced proliferation of allogeneic lymphocytes. Thus, the cells derived by these culture conditions had markers and functions analogous to immature myeloid DCs. Availability of feline DCs will enable investigation of their role in infectious disease and their potential therapeutic application.

Multiple Myeloma in Cats: Variable Presentation with Different Immunoglobulin Isotypes in Two Cats

Multiple myeloma was diagnosed in two cats with monoclonal hyperglobulinemia, proteinuria, and plasma cell proliferations in bone marrow. An immunoglobulin G-producing myeloma occurred in the vertebral bone marrow of one cat, and twice responded to surgical reduction followed by a combination of local irradiation and chemotherapy. The cats survival time was approximately 2 years. The other myeloma in a cat that presented with hypercalcemia and renal insufficieny involved visceral organs and produced a biclonal peak due to immunoglobulin A dimer formation on serum electrophoresis. This cats tumor did not respond to chemotherapy.