Homoharringtonine demonstrates a cytotoxic effect against triple-negative breast cancer cell lines and acts synergistically with paclitaxel.

The lack of targeted therapies for triple-negative breast cancer (TNBC) contributes to their high mortality rates and high risk of relapse compared to other subtypes of breast cancer. Most TNBCs (75%) have downregulated the expression of CREB3L1 (cAMP-responsive element binding protein 3 like 1), a transcription factor and metastasis suppressor that represses genes that promote cancer progression and metastasis. In this report, we screened an FDA-approved drug library and identified four drugs that were highly cytotoxic towards HCC1806 CREB3L1-deficient TNBC cells. These four drugs were: (1) palbociclib isethionate, a CDK4/6 inhibitor, (2) lanatocide C (also named isolanid), a Na+/K+-ATPase inhibitor, (3) cladribine, a nucleoside analog, and (4) homoharringtonine (also named omacetaxine mepesuccinate), a protein translation inhibitor. Homoharringtonine consistently showed the most cytotoxicity towards an additional six TNBC cell lines (BT549, HCC1395, HCC38, Hs578T, MDA-MB-157, MDA-MB-436), and several luminal A breast cancer cell lines (HCC1428, MCF7, T47D, ZR-75-1). All four drugs were then separately evaluated for possible synergy with the chemotherapy agents, doxorubicin (an anthracycline) and paclitaxel (a microtubule stabilizing agent). A strong synergy was observed using the combination of homoharringtonine and paclitaxel, with high cytotoxicity towards TNBC cells at lower concentrations than when each was used separately.

Reduced CREB3L1 expression in triple negative and luminal a breast cancer cells contributes to enhanced cell migration, anchorage-independent growth and metastasis.

Women with metastatic breast cancer have a disheartening 5-year survival rate of only 28%. CREB3L1 (cAMP-responsive element binding protein 3 like 1) is a metastasis suppressor that functions as a transcription factor, and in an estrogen-dependent model of rat breast cancer, it repressed the expression of genes that promote breast cancer progression and metastasis. In this report, we set out to determine the expression level of CREB3L1 across different human breast cancer subtypes and determine whether CREB3L1 functions as a metastasis suppressor, particularly in triple negative breast cancers (TNBCs). CREB3L1 expression was generally increased in luminal A, luminal B and HER2 breast cancers, but significantly reduced in a high proportion (75%) of TNBCs. Two luminal A (HCC1428, T47D) and two basal TNBC (HCC1806, HCC70) CREB3L1-deficient breast cancer cell lines were characterized as compared to their corresponding HA-CREB3L1-expressing counterparts. HA-CREB3L1 expression significantly reduced both cell migration and anchorage-independent growth in soft agar but had no impact on cell proliferation rates as compared to the CREB3L1-deficient parental cell lines. Restoration of CREB3L1 expression in HCC1806 cells was also sufficient to reduce mammary fat pad tumor formation and lung metastases in mouse xenograft models of breast cancer as compared to the parental HCC1806 cells. These results strongly support a metastasis suppressor role for CREB3L1 in human luminal A and TNBCs. Further, the ability to identify the subset of luminal A (7%) and TNBCs (75%) that are CREB3L1-deficient provides opportunities to stratify patients that would benefit from additional treatments to treat their more metastatic disease.

Impact of p85α Alterations in Cancer.

The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in the regulation of cell signaling, proliferation, survival, migration and vesicle trafficking in normal cells and is frequently deregulated in many cancers. The p85α protein is the most characterized regulatory subunit of the class IA PI3Ks, best known for its regulation of the p110-PI3K catalytic subunit. In this review, we will discuss the impact of p85α mutations or alterations in expression levels on the proteins p85α is known to bind and regulate. We will focus on alterations within the N-terminal half of p85α that primarily regulate Rab5 and some members of the Rho-family of GTPases, as well as those that regulate PTEN (phosphatase and tensin homologue deleted on chromosome 10), the enzyme that directly counteracts PI3K signaling. We highlight recent data, mapping the interaction surfaces of the PTEN⁻p85α breakpoint cluster region homology (BH) domain, which sheds new light on key residues in both proteins. As a multifunctional protein that binds and regulates many different proteins, p85α mutations at different sites have different impacts in cancer and would necessarily require distinct treatment strategies to be effective.

Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation.

The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.

A novel non-azole topical treatment reduces Malassezia numbers and associated dermatitis: a short term prospective, randomized, blinded and placebo-controlled trial in naturally infected dogs.

BACKGROUND: Malassezia yeast overgrowth on the skin is a common and often recurrent cause of dermatitis in dogs; it can be an exacerbating factor of atopic dermatitis. Anti-fungal drugs have been a standard treatment, but there is some concern that resistance may be evolving in a spectrum of Malassezia species. Safe, efficient and easy-to-use alternatives are needed. OBJECTIVES: To assess if a commercially available topical non-azole solution applied to paws affected by Malassezia-associated dermatitis (MAD), could ameliorate Malassezia numbers and associated signs over a short term (14 day) trial. ANIMALS: Eighteen dogs with MAD affecting at least two paws. METHODS: The study design was prospective, randomized, blinded and placebo-controlled, using a split-body protocol. Dogs were treated once daily with the test solution on one paw and placebo on the other. Dogs were examined at days 0 and 14 ± 3. The primary end-point was Malassezia numbers assessed cytologically. Secondary end-points were clinical scores for lesion severity and pruritus as assessed by a pruritus Visual Analog Scale (PVAS). Owner compliance and adverse effects were assessed. RESULTS: There was a statistically significant reduction in Malassezia numbers and clinical scores for paws treated with the test solution versus placebo. No statistical difference in PVAS was found. CONCLUSION: Daily topical application of the test solution was effective in reducing the Malassezia burden, as well as improving clinical scores in dogs with MAD of the paws. No adverse effects were reported and owners described the product as either "easy" or "very easy" to use.

Insight into the PTEN - p85α interaction and lipid binding properties of the p85α BH domain.

The phosphatidylinositol 3-kinase (PI3K) pathway plays a key role in regulating cell growth and cell survival and is frequently deregulated in cancer cells. p85α regulates the p110α lipid kinase, and also stabilizes and stimulates PTEN, the lipid phosphatase that downregulates this pathway. In this report, we determined that the p85α BH domain binds several phosphorylated phosphoinositide lipids, an interaction that could help localize p85α to membranes rich in these lipids. We also identified key residues responsible for mediating PTEN - p85α complex formation. Based on these experimental results, a docking model for the PTEN - p85α BH domain complex was developed that is consistent with the known binding interactions for both PTEN and p85α. This model involves extensive side-chain and peptide backbone contacts between both the PASE and C2 domains of PTEN with the p85α BH domains. The p85α BH domain residues shown to be important for PTEN binding were p85α residues E212, Q221, K225, R228 and H234. We also verified experimentally the importance of PTEN-E91 in mediating the interaction with the p85α BH domain. These results shed new light on the mechanism of PTEN regulation by p85α.

The role of PTEN - HCV core interaction in hepatitis C virus replication.

Hepatitis C virus (HCV) infection leads to severe liver diseases including hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumour suppressor, is frequently mutated or deleted in HCC tumors. PTEN has previously been demonstrated to inhibit HCV secretion. In this study, we determined the effects of PTEN on the other steps in HCV life cycle, including entry, translation, and replication. We showed that PTEN inhibits HCV entry through its lipid phosphatase activity. PTEN has no effect on HCV RNA translation. PTEN decreases HCV replication and the protein phosphatase activity of PTEN is essential for this function. PTEN interacts with the HCV core protein and requires R50 in domain I of HCV core and PTEN residues 1-185 for this interaction. This interaction is required for PTEN-mediated inhibition of HCV replication. This gives rise to a reduction in PTEN levels and intracellular lipid abundance, which may in turn regulate HCV replication. HCV core domain I protein increases the lipid phosphatase activity of PTEN in an in vitro assay, suggesting that HCV infection can also regulate PTEN. Taken together, our results demonstrated an important regulatory role of PTEN in the HCV life cycle.

Molecular characterization of breast cancer cell lines through multiple omic approaches.

BACKGROUND: Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated. METHODS: We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer. RESULTS: The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated in common or unique ways. CONCLUSIONS: These two new kinase or "Kin-OMIC" analyses add another dimension of important data about these frequently used breast cancer cell lines. This will assist researchers in selecting the most appropriate cell lines to use for breast cancer studies and provide context for the interpretation of the emerging results.

Epigenetic silencing of CREB3L1 by DNA methylation is associated with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers.

BACKGROUND: CREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the unfolded protein response, has recently been identified as a metastasis suppressor in both breast and bladder cancer. METHODS: Quantitative real time PCR (qPCR) and immunoblotting were used to determine the impact of histone deacetylation and DNA methylation inhibitors on CREB3L1 expression in breast cancer cell lines. Breast cancer cell lines and tumor samples were analyzed similarly, and CREB3L1 gene methylation was determined using sodium bisulfite conversion and DNA sequencing. Immunohistochemistry was used to determine nuclear versus cytoplasmic CREB3L1 protein. Large breast cancer database analyses were carried out to examine relationships between CREB3L1 gene methylation and mRNA expression in addition to CREB3L1 mRNA expression and prognosis. RESULTS: This study demonstrates that the low CREB3L1 expression previously seen in highly metastatic breast cancer cell lines is caused in part by epigenetic silencing. Treatment of several highly metastatic breast cancer cell lines that had low CREB3L1 expression with DNA methyltransferase and histone deacetylase inhibitors induced expression of CREB3L1, both mRNA and protein. In human breast tumors, CREB3L1 mRNA expression was upregulated in low and medium-grade tumors, most frequently of the luminal and HER2 amplified subtypes. In contrast, CREB3L1 expression was repressed in high-grade tumors, and its loss was most frequently associated with triple negative breast cancers (TNBCs). Importantly, bioinformatics analyses of tumor databases support these findings, with methylation of the CREB3L1 gene associated with TNBCs, and strongly negatively correlated with CREB3L1 mRNA expression. Decreased CREB3L1 mRNA expression was associated with increased tumor grade and reduced progression-free survival. An immunohistochemistry analysis revealed that low-grade breast tumors frequently had nuclear CREB3L1 protein, in contrast to the high-grade breast tumors in which CREB3L1 was cytoplasmic, suggesting that differential localization may also regulate CREB3L1 effectiveness in metastasis suppression. CONCLUSIONS: Our data further strengthens the role for CREB3L1 as a metastasis suppressor in breast cancer and demonstrates that epigenetic silencing is a major regulator of the loss of CREB3L1 expression. We also highlight that CREB3L1 expression is frequently altered in many cancer types suggesting that it could have a broader role in cancer progression and metastasis.

Identification of mutations in distinct regions of p85 alpha in urothelial cancer.

Bladder cancers commonly show genetic aberrations in the phosphatidylinositol 3-kinase signaling pathway. Here we have screened for mutations in PIK3R1, which encodes p85α, one of the regulatory subunits of PI3K. Two hundred and sixty-four bladder tumours and 41 bladder tumour cell lines were screened and 18 mutations were detected. Thirteen mutations were in C-terminal domains and are predicted to interfere with the interaction between p85α and p110α. Five mutations were in the BH domain of PIK3R1. This region has been implicated in p110α-independent roles of p85α, such as binding to and altering the activities of PTEN, Rab4 and Rab5. Expression of these mutant BH-p85α forms in mouse embryonic fibroblasts with p85α knockout indicated that all forms, except the truncation mutants, could bind and stabilize p110α but did not increase AKT phosphorylation, suggesting that BH mutations function independently of p110α. In a panel of 44 bladder tumour cell lines, 80% had reduced PIK3R1 mRNA expression relative to normal urothelial cells. This, along with mutation of PIK3R1, may alter BH domain functioning. Our findings suggest that mutant forms of p85α may play an oncogenic role in bladder cancer, not only via loss of ability to regulate p110α but also via altered function of the BH domain.

CREB3L1 is a metastasis suppressor that represses expression of genes regulating metastasis, invasion, and angiogenesis.

The unfolded protein response (UPR) is activated in response to hypoxia-induced stress such as in the tumor microenvironment. This study examined the role of CREB3L1 (cyclic AMP [cAMP]-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low-metastasis or nonmetastatic cell lines. When metastatic cells were transfected with CREB3L1, they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under nonadherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, not only did CREB3L1-expressing cells fail to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and chromatin immunoprecipitation with microarray technology (ChIP on Chip) analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. These data suggest that CREB3L1 plays an important role in suppressing tumorigenesis and that loss of expression is required for the development of a metastatic phenotype.

The signalling factor PI3K is a specific regulator of the clathrin-independent dynamin-dependent endocytosis of IL-2 receptors.

Receptor-mediated endocytosis is an essential process used by eukaryotic cells to internalise many molecules. Several clathrin-independent endocytic routes exist, but the molecular mechanism of each pathway remains to be uncovered. The present study focuses on a clathrin-independent dynamin-dependent pathway used by interleukin 2 receptors (IL-2R), essential players of the immune response. Ras-related C3 botulinum toxin substrate (Rac1) and its targets, the p21-activated kinases (Pak), are specific regulators of this pathway, acting on cortactin and actin polymerization. The present study reveals a dual and specific role of phosphatidylinositol 3-kinase (PI3K) in IL-2R endocytosis. Inhibition of the catalytic activity of PI3K strongly affects IL-2R endocytosis, in contrast to transferrin (Tf) uptake, a marker of the clathrin-mediated pathway. Moreover, Vav2, a GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved in IL-2R entry. The second action of PI3K is through its regulatory subunit, p85α, which binds to and recruits Rac1 during IL-2R internalisation. Indeed, the overexpression of a p85α mutant missing the Rac1 binding motif leads to the specific inhibition of IL-2R endocytosis. The inhibitory effect of this p85α mutant could be rescued by the overexpression of either Rac1 or the active form of Pak, indicating that p85α acts upstream of the Rac1-Pak cascade. Finally, biochemical and fluorescent microscopy techniques reveal an interaction between p85α, Rac1 and IL-2R that is enhanced by IL-2. In summary, our results indicate a key role of class I PI3K in IL-2R endocytosis that creates a link with IL-2 signalling.

Multiple roles for the p85α isoform in the regulation and function of PI3K signalling and receptor trafficking.

The p85α protein is best known as the regulatory subunit of class 1A PI3Ks (phosphoinositide 3-kinases) through its interaction, stabilization and repression of p110-PI3K catalytic subunits. PI3Ks play multiple roles in the regulation of cell survival, signalling, proliferation, migration and vesicle trafficking. The present review will focus on p85α, with special emphasis on its important roles in the regulation of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and Rab5 functions. The phosphatidylinositol-3-phosphatase PTEN directly counteracts PI3K signalling through dephosphorylation of PI3K lipid products. Thus the balance of p85α-p110 and p85α-PTEN complexes determines the signalling output of the PI3K/PTEN pathway, and under conditions of reduced p85α levels, the p85α-PTEN complex is selectively reduced, promoting PI3K signalling. Rab5 GTPases are important during the endocytosis, intracellular trafficking and degradation of activated receptor complexes. The p85α protein helps switch off Rab5, and if defective in this p85α function, results in sustained activated receptor tyrosine kinase signalling and cell transformation through disrupted receptor trafficking. The central role for p85α in the regulation of PTEN and Rab5 has widened the scope of p85α functions to include integration of PI3K activation (p110-mediated), deactivation (PTEN-mediated) and receptor trafficking/signalling (Rab5-mediated) functions, all with key roles in maintaining cellular homoeostasis.

Ovarian cancer stem cell-like side populations are enriched following chemotherapy and overexpress EZH2.

Platinum-based chemotherapy, with cytoreductive surgery, is the cornerstone of treatment of advanced ovarian cancer; however, acquired drug resistance is a major clinical obstacle. It has been proposed that subpopulations of tumor cells with stem cell-like properties, such as so-called side populations (SP) that overexpress ABC drug transporters, can sustain the growth of drug-resistant tumor cells, leading to tumor recurrence following chemotherapy. The histone methyltransferase EZH2 is a key component of the polycomb-repressive complex 2 required for maintenance of a stem cell state, and overexpression has been implicated in drug resistance and shorter survival of ovarian cancer patients. We observed higher percentage SP in ascites from patients that have relapsed following chemotherapy compared with chemonaive patients, consistent with selection for this subpopulation during platinum-based chemotherapy. Furthermore, ABCB1 (P-glycoprotein) and EZH2 are consistently overexpressed in SP compared with non-SP from patients' tumor cells. The siRNA knockdown of EZH2 leads to loss of SP in ovarian tumor models, reduced anchorage-independent growth, and reduced tumor growth in vivo. Together, these data support a key role for EZH2 in the maintenance of a drug-resistant, tumor-sustaining subpopulation of cells in ovarian cancers undergoing chemotherapy. As such, EZH2 is an important target for anticancer drug development.

Anti-inflammatory therapy by intravenous delivery of non-heparan sulfate-binding CXCL12.

Interaction between chemokines and heparan sulfate (HS) is essential for leukocyte recruitment during inflammation. Previous studies have shown that a non-HS-binding mutant form of the inflammatory chemokine CCL7 can block inflammation produced by wild-type chemokines. This study examined the anti-inflammatory mechanism of a non-HS-binding mutant of the homeostatic chemokine CXCL12. Initial experiments demonstrated that mutant CXCL12 was an effective CXCR4 agonist. However, this mutant chemokine failed to promote transendothelial migration in vitro and inhibited the haptotactic response to wild-type CCL7, CXCL12, and CXCL8, and naturally occurring chemoattractants in synovial fluid from the rheumatoid synovium, including CCL2, CCL7, and CXCL8. Notably, intravenous administration of mutant CXCL12 also inhibited the recruitment of leukocytes to murine air pouches filled with wild-type CXCL12. Following intravenous administration, wild-type CXCL12 was cleared from the circulation rapidly, while the mutant chemokine persisted for >24 h. Chronic exposure to mutant CXCL12 in the circulation reduced leukocyte-surface expression of CXCR4, reduced the chemotactic response of these cells to CXCL12, and inhibited normal chemokine-mediated induction of adhesion between the alpha4beta1 integrin, VLA-4, and VCAM-1. These data demonstrate that systemic administration of non-HS-binding variants of CXCL12 can mediate a powerful anti-inflammatory effect through chemokine receptor desensitization.

Inhibition of CXCR4-mediated breast cancer metastasis: a potential role for heparinoids?

PURPOSE: The pattern of breast cancer metastasis may be determined by interactions between CXCR4 on breast cancer cells and CXCL12 within normal tissues. Glycosaminoglycans bind chemokines for presentation to responsive cells. This study was designed to test the hypothesis that soluble heparinoid glycosaminoglycan molecules can disrupt the normal response to CXCL12, thereby reducing the metastasis of CXCR4-expressing cancer cells. EXPERIMENTAL DESIGN: Inhibition of the response of CXCR4-expressing Chinese hamster ovary cells to CXCL12 was assessed by measurement of calcium flux and chemotaxis. Radioligand binding was also assessed to quantify the potential of soluble heparinoids to prevent specific receptor ligation. The human breast cancer cell line MDA-MB-231 and a range of sublines were assessed for their sensitivity to heparinoid-mediated inhibition of chemotaxis. A model of hematogenous breast cancer metastasis was established, and the potential of clinically relevant doses of heparinoids to inhibit CXCL12 presentation and metastatic disease was assessed. RESULTS: Unfractionated heparin and the low-molecular-weight heparin tinzaparin inhibited receptor ligation and the response of CXCR4-expressing Chinese hamster ovary cells and human breast cancer cell lines to CXCL12. Heparin also removed CXCL12 from its normal site of expression on the surface of parenchymal cells in the murine lung. Both heparin and two clinically relevant dose regimens of tinzaparin reduced hematogenous metastatic spread of human breast cancer cells to the lung in a murine model. CONCLUSIONS: Clinically relevant concentrations of tinzaparin inhibit the interaction between CXCL12 and CXCR4 and may be useful to prevent chemokine-driven breast cancer metastasis.

An apparent paradox: chemokine receptor agonists can be used for anti-inflammatory therapy.

Inflammation plays an important role in a wide range of human diseases. Chemokines are a group of proteins which control the migration and activation of the immune cells involved in all aspects of the inflammatory response. Chemokines bind to specific receptors of the seven-transmembrane spanning type on target leukocytes and also bind to cell-surface glycosaminoglycans (GAG). Leukocytes express a range of chemokine receptors which can cross-desensitise each other, potentially allowing a single chemokine receptor agonist to desensitise all the chemokine receptors on a cell. If an appropriate single receptor agonist is engineered to be non-chemotactic itself, then a treated cell will lose the potential to migrate in response to chemokines towards any developing site of inflammation. A non-GAG-binding but receptor agonistic form of the chemokine CCL7 can inhibit leukocyte recruitment in response to a diverse range of chemokines in vitro and in vivo. We hypothesise that this modified chemokine mediates its effect by inducing homologous and heterologous receptor desensitisation and further propose that other suitable candidates could include agonistic chemokine receptor-specific antibodies or small molecule chemokine receptor agonists. Hence, an appropriate chemokine receptor agonist could be used to inhibit multiple chemokine receptors, thereby producing a powerful and robust anti-inflammatory effect. This review considers the mechanisms leading to chemokine receptor desensitisation and discusses the potential to develop a new class of anti-inflammatory agents based on targeted stimulation of chemokine receptors.

Solitary plasmacytoma of bone in two successfully treated cats.

This is the first report of feline solitary plasmacytoma of bone. We describe the clinical, clinico-pathological, radiographic and pathological findings of two successfully treated cats with long-term follow-up. The first case presented with spinal pain and neurological deficits. Radiographs demonstrated sclerosis of lumbar vertebra L6 and a myelogram confirmed interference to flow of contrast in the L4-7 region. A biopsy of L6 revealed neoplastic plasma cell infiltration. There was no evidence of paraproteinaemia on serum protein electrophoresis. The cat underwent hypofractionated megavoltage radiotherapy. Clinical signs resolved completely and 4 years after diagnosis the cat remains well and has no electrophoretically detectable paraproteinaemia. The second case presented with neurological deficits of the tail and spinal radiographs revealed extensive osteolysis of the sacrum. A biopsy of sacral bone demonstrated neoplastic plasma cell infiltration. The animal was normoglobulinaemic. The cat improved clinically with induction chemotherapy (melphalan and methylprednisolone). The same chemotherapeutics were continued at maintenance doses for 4.3 years, at which time there was recurrence of neurological deficits and a palpable sacral mass. Cytological examination of a fine needle aspirate confirmed recurrence of plasma cell neoplasia. A low concentration monoclonal paraproteinaemia was detected. Vincristine was administered resulting in resolution of neurological deficits and a palpably smaller sacral mass. Eighteen months into vincristine therapy, there was recurrence of clinical signs and the cat was euthanased, more than 6 years after the initial diagnosis.

Myeloma-related disorders in cats commonly present as extramedullary neoplasms in contrast to myeloma in human patients: 24 cases with clinical follow-up.

BACKGROUND: Myeloma-related disorders (MRD) are rare neoplasms of plasma cells. Published case reports describe a diversity of clinical presentations with confusing terminology and diagnostic criteria as a consequence of the assumption that MRD in cats are analogous to those in dogs or humans. OBJECTIVE: The aim of the study was to describe clinical, clinicopathologic and imaging findings, response to treatment, survival and possible associations with other diseases or vaccination in a large case series. A priori hypotheses were that cats with MRD commonly present with extramedullary involvement and uncommonly have radiographic bone lesions, in contrast to human patients. ANIMALS: Twenty-four cats with MRD confirmed by cytology or histopathology and immunohistochemistry. METHOD: A multicenter retrospective study was performed. RESULTS: Two types of clinical presentation were observed. The first group (n = 17) had neoplasia involving abdominal organs, bone marrow, or both. All developed systemic clinical signs and paraproteinemia. Five of 7 cats that received chemotherapy improved clinically or had decreased serum globulin concentration (median survival, 12.3 months; range, 8.5-22 months). The second group comprised 7 cats with skin masses, 2 of which were paraproteinemic and developed rapidly worsening systemic signs. In cats without systemic signs, excision of the skin masses appeared to be associated with prolonged survival (up to 2.4 years). Cats with MRD commonly presented with extramedullary involvement (67%), versus humans with MRD (5%) (P < .001), and uncommonly presented with radiographic bone lesions (8%) versus humans with MRD (80%) (P < .001). CONCLUSIONS: Radiographic bone lesions are uncommon in cats with MRD and extramedullary presentation is common, relative to human myeloma.

High serum troponin I concentration as a marker of severe myocardial damage in a case of suspected exertional heatstroke in a dog.

A young, overweight dog presented with sudden onset lethargy and collapse following exercise in warm environmental conditions. Investigations revealed systolic hypotension, multiform ventricular premature complexes, irregular myocardial echogenicity with poor left ventricular systolic function and a markedly elevated troponin cTnI (180ng/mL, reference range <0.3ng/mL) consistent with severe myocyte damage. Infectious causes of myocarditis were ruled out on the basis of serological and polymerase chain reaction blood tests. Exercise-induced malignant hyperthermia was excluded from the history, an exercise tolerance test and genetic testing for the RYR1 V547A mutation. The diagnosis was myocardial damage secondary to suspected exertional heatstroke, from which the dog recovered uneventfully over a number of weeks and serum troponin normalised. This is the first case report in any species including man, documenting high troponin as a marker of severe myocardial damage following suspected heatstroke.