Evaluation of responses to immunosuppressive therapy in dogs with suspected non-regenerative immune-mediated anaemia: 11 cases (2012-2018).

OBJECTIVES: We aimed to determine the response time to immunosuppressive therapy and time required to achieve a 5% increase in haematocrit among dogs with non-regenerative immune-mediated anaemia. MATERIALS AND METHODS: Client-owned dogs diagnosed with non-regenerative immune-mediated anaemia in Hokkaido University Veterinary Teaching Hospital between December 2012 and May 2018 were enrolled. The first treatment regimen included prednisolone (2 mg/kg/day) and ciclosporin (up to 10 mg/kg/day) for 8 weeks. Dogs that did not respond to the first regimen proceeded to the second regimen comprising prednisolone and mycophenolate mofetil (15 mg/kg, twice a day). Reticulocyte count and haematocrit were monitored every 1 to 2 weeks. Treatment response was defined as an absolute reticulocyte count more than 60×103 /µL or increasing haematocrit. RESULTS: During the study period, 23 dogs fulfilled the inclusion criteria for non-regenerative immune-mediated anaemia. Twelve dogs were excluded from this study for various reasons and response to therapy was evaluated in the remaining 11 dogs. Treatment responses were observed in 8 of 11 dogs, and the median time to response was 39.5 days (range 8 to 92 days). Two responders were unable to continue the first treatment regimen and were switched to the second regimen owing to anorexia and nausea, possibly induced by ciclosporin; withdrawal of ciclosporin improved their symptoms. The time required to achieve a 5% increase in haematocrit was assessed in the other six dogs, with a median of 55.5 days (range 8 to 135 days). CLINICAL SIGNIFICANCE: Here we report the response to a standardised treatment protocol in dogs with non-regenerative immune-mediated anaemia. Knowledge of potential side effects and expected therapeutic outcomes may be of use for veterinary practitioners treating this condition.

Olsalazine inhibits cell proliferation and DNA methylation in canine lymphoid tumor cell lines.

Abnormal DNA methylation is involved in the initiation and progression of lymphoid tumors. Hence, DNA demethylating agents are promising candidate drugs for chemotherapy against these tumors. The salicylic acid derived anti-inflammatory agent, olsalazine, reportedly suppresses DNA methyltransferase in human cells and has the potential to be clinically applied as a DNA demethylating agent. In this study, we investigated the effects of olsalazine on cell proliferation and DNA methylation using canine lymphoid tumor cell lines (CLBL-1, GL-1, and UL-1). Treatment with olsalazine led to significant cell growth inhibition and increased the apoptotic rate in all three cell lines. Treatment with olsalazine reduced the total amount of 5-methylcytosine in genomic DNA, as assessed by enzyme-linked immunosorbent assay. Genome-wide analysis of DNA methylation revealed that 1,801 to 5,626 CpG sites showed decreased DNA methylation levels in three cell lines, including the promoter regions of ADAM23, FES, and CREB3L1 genes. The outcomes of the present study demonstrate that a DNA demethylating agent olsalazine, inhibits cell proliferation and DNA methylation in canine lymphoid tumor cells, suggesting that it can be a candidate drug for the treatment of lymphoid tumors in dogs.

Genome-wide DNA methylation analysis identifies promoter hypermethylation in canine malignant melanoma.

Canine malignant melanoma is a common cancer with a high mortality rate. Although previous studies have evaluated various aspects of this tumour, the exact mechanism of tumourigenesis remains unknown. Epigenetic mechanisms, such as DNA methylation, have recently gained attention as aetiological factors for neoplasia in humans. This study aimed to analyse genome-wide DNA methylation patterns in canine malignant melanoma based on next-generation sequencing data. A total of 76,213 CpG sites, including 29,482 sites in CpG islands (CGIs), were analysed using next-generation sequencing of methylation-specific signatures, obtained by sequential digestion with enzymes, to compare normal oral mucosal samples from four healthy dogs, four canine melanoma cell lines (3 oral cavity and 1 skin), and five clinical samples of oral canine melanoma. Malignant melanoma showed increased methylation at thousands of normally unmethylated CpG sites in CGIs and decreased methylation at normally methylated CpG sites in non-CGIs. Interestingly, the promoter regions of 81-393 genes were hypermethylated; 23 of these genes were present in all melanoma cell lines and melanoma clinical samples. Among these 23 genes, six genes with "sequence-specific DNA binding" annotation were significantly enriched, including three Homeobox genes-HMX2, TLX2, and HOXA9-that may be involved in the tumourigenesis of canine malignant melanoma. This study revealed widespread alterations in DNA methylation and a large number of hypermethylated genes in canine malignant melanoma.

Peptidylarginine deiminase is involved in maintaining the cornified oral mucosa of rats.

BACKGROUND AND OBJECTIVE: Epithelial cells derived from different regions exhibit marked differences in their differentiation capacity, allowing them to provide a suitable protective barrier. We aimed to clarify the role of peptidylarginine deiminase (PAD) in modifying the key epidermal proteins filaggrin (FLG) and keratin 1 (K1) during stratification of the rat palate and buccal mucosa. MATERIAL AND METHODS: We performed immunofluorescence, immunoblotting, PAD activity assays and 2-dimensional electrophoresis, and developed an organotypic culture model. RESULTS: PAD1 expression was highest in the palate, whereas PAD2, PAD3 and PAD4 expression was highest in the skin, suggesting the tissue-specific expression of PAD isozymes that leads to differences in calcium dependency. Immunoblotting showed that the FLG monomer, as well as its degradation products and precursor (proFLG), were most abundantly expressed in the skin but had low expression in the palate, whereas only faint proFLG expression was detected in the buccal mucosa. FLG and K1 were colocalized with PAD1 and were likely to be citrullinated in the cornified layers of the skin; this colocalization was not detected on the palatal surface, and dot-like presence of proFLG that might be citrullinated and that of PAD1 were found in the granules of the palate. Organotypic models derived from the rat palate revealed that PAD inhibition reduced the breakdown of FLG, increased its association with K1 together with epithelial compaction, and decreased permeability in a dye permeability assay. Conversely, PAD stimulation had the opposite effects. CONCLUSION: Citrullination is likely a protein modification that plays an important role in maintaining the structure and function of oral cornified mucosa in a way that is distinctly different from that of the skin.

Dynamic changes in DNA methylation patterns in canine lymphoma cell lines demonstrated by genome-wide quantitative DNA methylation analysis.

DNA methylation is the conversion of cytosine to 5-methylcytosine, leading to changes in the interactions between DNA and proteins. Methylation of cytosine-guanine (CpG) islands (CGIs) is associated with gene expression silencing of the involved promoter. Although studies focussing on global changes or a few single loci in DNA methylation have been performed in dogs with certain diseases, genome-wide analysis of DNA methylation is required to prospectively identify specific regions with DNA methylation change. The hypothesis of this study was that next-generation sequencing with methylation-specific signatures created by sequential digestion of genomic DNA with SmaI and XmaI enzymes can provide quantitative information on methylation levels. Using blood from healthy dogs and cells obtained from canine lymphoma cell lines, approximately 100,000CpG sites across the dog genome were analysed with the novel method established in this study. CpG sites in CGIs broadly were shown to be either methylated or unmethylated in normal blood, while CpG sites not within CpG islands (NCGIs) were largely methylated. Thousands of CpG sites in lymphoma cell lines were found to gain methylation at normally unmethylated CGI sites and lose methylation at normally methylated NCGI sites. These hypermethylated CpG sites are located at promoter regions of hundreds of genes, such as TWIST2 and TLX3. In addition, genes annotated with 'Homeobox' and 'DNA-binding' characteristics have hypermethylated CpG sites in their promoter CGIs. Genome-wide quantitative DNA methylation analysis is a sensitive method that is likely to be suitable for studies of DNA methylation changes in cancer, as well as other common diseases in dogs.

Localization of Toll-like Receptor (TLR) 2 and TLR4 mRNA in the Colorectal Mucosa of Miniature Dachshunds with Inflammatory Colorectal Polyps.

Inflammatory colorectal polyps (ICRPs) are characterized by the formation of multiple or solitary polyps with marked neutrophil infiltration in the colorectal area, and are speculated to be a novel form of breed-specific canine idiopathic inflammatory bowel disease (IBD). In human IBD, toll-like receptor (TLR) 2 and TLR4 have been reported to be involved in the pathogenesis of the disease. The aim of this study was to evaluate the expression of TLR2 and TLR4 mRNA in the colorectal mucosa of dogs with ICRPs by in-situ hybridization using an RNAscope assay. Samples of inflamed colorectal mucosa (n = 5) and non-inflamed mucosa (n = 5) from miniature dachshunds (MDs) with ICRPs and colonic mucosa from healthy beagles (n = 5) were examined. TLR2 and TLR4 hybridization signals were localized to the colorectal epithelium, inflammatory cells and fibroblasts in the inflamed colorectal mucosa of affected dogs. The signals were significantly greater in inflamed colorectal epithelium compared with non-inflamed epithelium of MDs with ICRPs and healthy beagles (P <0.05). These results suggest that increased expression of TLR2 and TLR4 mRNA in the inflamed colorectal mucosa results from not only inflammatory cell infiltration, but also the upregulation of TLR2 and TLR4 mRNA in the colonic epithelium.

A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome.

Genetic changes are infrequent in acute myeloid leukemia (AML) compared with other malignancies and often involve epigenetic regulators, suggesting that an altered epigenome may underlie AML biology and outcomes. In 96 AML cases including 65 pilot samples selected for cured/not-cured, we found higher CpG island (CGI) promoter methylation in cured patients. Expanded genome-wide digital restriction enzyme analysis of methylation data revealed a CGI methylator phenotype independent of IDH1/2 mutations we term AML-CGI methylator phenotype (CIMP) (A-CIMP+). A-CIMP was associated with longer overall survival (OS) in this data set (median OS, years: A-CIMP+=not reached, CIMP-=1.17; P=0.08). For validation we used 194 samples from The Cancer Genome Atlas interrogated with Illumina 450k methylation arrays where we confirmed longer OS in A-CIMP (median OS, years: A-CIMP+=2.34, A-CIMP-=1.00; P=0.01). Hypermethylation in A-CIMP+ favored CGIs (OR: CGI/non-CGI=5.21), and while A-CIMP+ was enriched in CEBPA (P=0.002) and WT1 mutations (P=0.02), 70% of cases lacked either mutation. Hypermethylated genes in A-CIMP+ function in pluripotency maintenance, and a gene expression signature of A-CIMP was associated with outcomes in multiple data sets. We conclude that CIMP in AML cannot be explained solely by gene mutations (for example, IDH1/2, TET2), and that curability in A-CIMP+ AML should be validated prospectively.

Association of caspase-14 and filaggrin expression with keratinization of the oral mucosa and reconstruction culture rat models.

BACKGROUND AND OBJECTIVE: Keratinization of the oral mucosa, such as the gingiva, has been shown to be important for periodontal health. Caspase-14 is a protease that plays a role in keratinization of the epidermis. The objective of this study was to investigate whether the expression of caspase-14 is intimately linked with keratinization and to examine the effect of the main component of green tea on the improvement of keratinization in rat oral mucosal preparations. MATERIAL AND METHODS: Histological and immunohistochemical analyses and quantitative mRNA measurements of caspase-14 and its substrate filaggrin were performed using different types of rat epithelial tissue and organotypic reconstruction culture models derived from epithelial cells and fibroblasts taken from the rat oral mucosa. RESULTS: In the skin, palate, buccal mucosa and esophagus, the degree of keratinization appeared to be associated with expression of cytokeratin 10. The relative protein and mRNA expression levels of caspase-14 and filaggrin were consistent with the degree of keratinization in the following order: skin > palate > buccal mucosa > esophagus. The culture models of palatal and buccal mucosa retained a stratified epithelial structure. Expression of caspase-14 appeared to be stronger in the palatal model than in the buccal model. Remarkably, epigallocatechin-3-gallate (EGCG) improved the localization of cytokeratins and increased the expression of caspase-14 and filaggrin. This expression was more intense in the palatal model than in the buccal model, indicating that both models maintain the intrinsic properties of keratinization of the mucosa from where the cultured cells were derived. CONCLUSIONS: These results suggest that keratinization is closely associated with expression of caspase-14 and filaggrin. Our reconstruction models are promising tools for drug evaluation and show that EGCG is beneficial for improving both keratinization and expression of the linked protease in the oral mucosa.

Involvement of IP3-receptor activation in endothelin-1-induced Ca(2+) influx in rat pulmonary small artery.

We examined the endothelin-1 (ET-1)-induced increase in the intracellular free Ca(2+) concentration ([Ca(2+)]i) in fura-2-loaded rat pulmonary small arteries. ET-1 (30 nM) elicited a long-lasting increase in [Ca(2+)]i in physiological salt solution (PSS). In subsequent experiments, arteries were pretreated with BQ-788, an ETB-specific blocker, to allow us to focus on responses mediated via the ETA receptor, the existence of which was confirmed by immunohistochemistry. In Ca(2+)-free PSS, ET-1 evoked a small transient increase in [Ca(2+)]i, indicating Ca(2+) release from the SR (sarcoplasmic reticulum). After a switch to PSS (containing 2mM CaCl2), ET-1 elicited a long-lasting increase in [Ca(2+)]i that was not inhibited by 1 µM nicardipine, an L-type Ca(2+)-channel inhibitor, suggesting involvement of a Ca(2+)-influx pathway independent of that channel. In arteries preincubated with 30 µM cyclopiazonic acid (CPA) or 2 µM thapsigargin (TG), the ET-1-induced Ca(2+)-release was greatly reduced, and the induced Ca(2+)-influx was attenuated. U-73122, a phospholipase C (PLC) inhibitor, had inhibitory effects similar to those of CPA and TG on the ET-1-induced Ca(2+)-release and Ca(2+)-influx, whereas U-73343, an inactive analogue of U-73122, had no such effects. Two putative membrane-permeable IP3-receptor blockers, 2-aminoethoxydiphenyl borate (2APB, 50 µM) and Xestospongin C (20 µM), (a) almost completely inhibited the ET-1-induced Ca(2+)-release and Ca(2+)-influx, and (b) reduced the ET-1-induced contraction. These results indicate that in rat pulmonary small arteries, ET-1 induces receptor-operated Ca(2+) influx via the ETA receptor, and that this influx interacts with InsP3-receptor activation.

Inhibition of T-type calcium channels and hydrogen sulfide-forming enzyme reverses paclitaxel-evoked neuropathic hyperalgesia in rats.

Hydrogen sulfide (H2S), a gasotransmitter, facilitates pain sensation by targeting Ca(v)3.2 T-type calcium channels. The H2S/Ca(v)3.2 pathway appears to play a role in the maintenance of surgically evoked neuropathic pain. Given evidence that chemotherapy-induced neuropathic pain is blocked by ethosuximide, known to block T-type calcium channels, we examined if more selective T-type calcium channel blockers and also inhibitors of cystathionine-γ-lyase (CSE), a major H2S-forming enzyme in the peripheral tissue, are capable of reversing the neuropathic pain evoked by paclitaxel, an anti-cancer drug. It was first demonstrated that T-type calcium channel blockers, NNC 55-0396, known to inhibit Ca(v)3.1, and mibefradil inhibited T-type currents in Ca(v)3.2-transfected HEK293 cells. Repeated systemic administration of paclitaxel caused delayed development of mechanical hyperalgesia, which was reversed by single intraplantar administration of NNC 55-0396 or mibefradil, and by silencing of Ca(v)3.2 by antisense oligodeoxynucleotides. Systemic administration of dl-propargylglycine and ß-cyanoalanine, irreversible and reversible inhibitors of CSE, respectively, also abolished the established neuropathic hyperalgesia. In the paclitaxel-treated rats, upregulation of Ca(v)3.2 and CSE at protein levels was not detected in the dorsal root ganglia (DRG), spinal cord or peripheral tissues including the hindpaws, whereas H(2)S content in hindpaw tissues was significantly elevated. Together, our study demonstrates the effectiveness of NNC 55-0396 in inhibiting Ca(v)3.2, and then suggests that paclitaxel-evoked neuropathic pain might involve the enhanced activity of T-type calcium channels and/or CSE in rats, but not upregulation of Ca(v)3.2 and CSE at protein levels, differing from the previous evidence for the neuropathic pain model induced by spinal nerve cutting in which Ca(v)3.2 was dramatically upregulated in DRG.

Increase in minimal residual disease in peripheral blood before clinical relapse in dogs with lymphoma that achieved complete remission after chemotherapy.

BACKGROUND: We developed previously a minimal residual disease (MRD) monitoring system in dogs with lymphoma by exploring a highly sensitive real-time PCR system. OBJECTIVES: To identify the change in MRD before clinical relapse in dogs with lymphoma that achieved complete remission after chemotherapy. ANIMALS: Twenty dogs with multicentric high-grade B-cell lymphoma. METHODS: MRD levels in peripheral blood mononuclear cells (PBMCs) were measured by real-time PCR amplifying the rearranged immunoglobulin heavy chain gene. MRD measurement and clinical assessment were performed every 2-4 weeks for 28-601 days after completion of chemotherapy. An increase in MRD was defined as an increase by more than 0.5, calculated by log10 [copy number of MRD per 105 PBMCs], based on the uncertainty level observed in a canine lymphoma cell line. RESULTS: During the follow-up period, 15 dogs relapsed in 28-320 days (median, 120 days) after completion of chemotherapy. An increase in MRD was detected 2 weeks or more before relapse in 14 of the 15 dogs, but an increase in MRD before relapse could not be detected in the remaining 1 dog. The time from increased MRD to clinical relapse was 0-63 days (median, 42 days). In contrast, no increase in MRD was detected in 5 dogs that did not experience clinical relapse. CONCLUSION AND CLINICAL IMPORTANCE: An increase in MRD can be detected before clinical relapse in dogs with lymphoma. Application of early reinduction therapy based on an increase in MRD before clinical relapse may improve treatment outcome in canine lymphoma.

Evaluation of cytoreductive efficacy of vincristine, cyclophosphamide, and Doxorubicin in dogs with lymphoma by measuring the number of neoplastic lymphoid cells with real-time polymerase chain reaction.

BACKGROUND: The cytoreductive efficacy of the individual components of multidrug chemotherapy for canine lymphoma is difficult to evaluate after complete remission. OBJECTIVES: To compare the cytoreductive efficacy of vincristine (VCR), cyclophosphamide (CPA), and doxorubicin (DXR) in dogs that received a 6-month modified version of the University of Wisconsin-Madison chemotherapy protocol (UW-25). ANIMALS: Twenty-nine dogs with high-grade B-cell multicentric lymphoma. METHODS: Rearranged immunoglobulin heavy chain gene fragments from lymphoma cells were amplified by polymerase chain reaction (PCR) and sequenced to prepare clone-specific primers and probes for real-time PCR. The number of lymphoma cells in peripheral blood was measured from diagnosis to week 11 of UW-25. RESULTS: The number of lymphoma cells after the 1st administration of VCR, CPA, and DXR in weeks 1-4 was decreased in 29/29 (100%), 15/29 (51.7%), and 26/27 (96.3%) dogs, respectively. The cytoreductive efficacy of CPA was less than that of VCR and DXR. VCR, CPA, and DXR administered in weeks 6-9 were effective in 5/26 (19.2%), 5/20 (25.0%), and 14/19 (73.7%) dogs, respectively, indicating the sustained cytoreductive efficacy of DXR. CPA nonresponders were heavier and exhibited a shorter 1st remission than CPA responders. CONCLUSION AND CLINICAL IMPORTANCE: When using UW-25 for treatment of canine lymphoma, CPA was found to have less cytoreductive efficacy than VCR and DXR. Real-time PCR-based quantification of tumor cells is an objective marker of the efficacy of chemotherapeutic agents.

Clinical outcomes of the placement of a drug-eluting stent with the use of Rotablator™ in dialytic patients: in comparison with non-dialytic patients.

Much remains unknown about the clinical outcomes of drug-eluting stent (DES) implantations following ablation by the Rotablator™ in dialytic patients. The purpose of this study was to examine these outcomes. Of the 43 patients who underwent elective DES implantation following ablation with the Rotablator™-17 lesions on 12 dialytic patients were designated to Group H and 54 lesions on 31 non-dialytic patients were designated to Group N. QCA data, the rate of restenosis and the avoidance rate of cardiac events at the chronic phase (920 ± 446 days) were studied. The age of patients in Group N was significantly older (60 ± 8 years old in Group H vs. 73 ± 7 in Group N, p < 0.05). The rate of DM complication was significantly higher in Group H (75.0% in Group H vs. 32.0% in Group N, p < 0.05). The late loss was 1.18 ± 0.97 mm in Group H and 0.38 ± 0.53 mm in Group N, or significantly greater in Group H (p < 0.05). The restenosis rate was also higher in group H. (42.9% in Group H and 6.0% in Group N). The avoidance rate of cardiac events at the chronic phase was significantly lower in Group H. The study suggested that in patients who received DES with the use of the Rotablator™, clinical outcomes were poorer in dialytic patients than in non-dialytic patients.

Monitoring of minimal residual disease (MRD) after multidrug chemotherapy and its correlation to outcome in dogs with lymphoma: a proof-of-concept pilot study.

BACKGROUND: Tumor cell burden in dogs with lymphoma cannot be assessed accurately by diagnostic evaluation during clinical complete remission (CR). Recent advances in polymerase chain reaction (PCR)-based methods enabled us to quantify minimal residual disease (MRD) in canine lymphoma. HYPOTHESIS/OBJECTIVES: To quantify MRD in dogs with lymphoma treated with multidrug chemotherapy and to correlate it with remission duration after chemotherapy. ANIMALS: Seventeen dogs with lymphoma that achieved CR by multidrug chemotherapy. METHODS: Rearranged immunoglobulin heavy chain or T-cell receptor gamma chain gene fragments from lymphoma cells were PCR amplified and sequenced to prepare clone-specific primers and probes for real-time PCR to quantify MRD. MRD in the peripheral blood was monitored during and at the end of a 25-week multidrug chemotherapy protocol. Correlation between MRD at the end of chemotherapy and remission duration after chemotherapy was analyzed. RESULTS: MRD gradually decreased after initiation of multidrug chemotherapy, reached a nadir as low as <0.019-1.0 cells/microL at weeks 4-17, and remained low or slightly increased until week 25. MRD at the end of chemotherapy was negatively correlated with remission duration from the end of chemotherapy to relapse. CONCLUSION AND CLINICAL IMPORTANCE: MRD could be an objective marker to indicate tumor cell burden in dogs with lymphoma even in clinical CR. MRD at the end of chemotherapy could be a prognostic factor to predict remission duration after chemotherapy.

Differences in kinematics of single leg squatting between anterior cruciate ligament-injured patients and healthy controls.

Seventy to eighty percent of all anterior cruciate ligament (ACL) injuries are due to non-contact injury mechanisms. It has been reported that the majority of injuries due to single leg landing come from valgus positioning of the lower leg. Preventing valgus positioning during single leg landing is expected to help reduce the number of ACL injuries. We found that many ACL-deficient patients cannot perform stable single leg squatting. Therefore, we performed 3D motion analysis of the single-legged half squat for ACL-injured patients to evaluate its significance as a risk factor for ACL injuries. We evaluated the relative angles between the body, thigh, and lower leg using an electromagnetic device during single leg half squatting performed by 63 ACL-injured patients (32 males, 31 females) the day before ACL reconstruction and by 26 healthy control subjects with no knee problems. The uninjured leg of ACL-injured male subjects demonstrated significantly less external knee rotation than that of the dominant leg of the male control. The uninjured leg of ACL-injured female subjects demonstrated significantly more external hip rotation and knee flexion and less hip flexion than that of the dominant leg of the female control. Comparing injured and uninjured legs, the injured leg of male subjects demonstrated significantly less external knee and hip rotation, less knee flexion, and more knee varus than that of the uninjured leg of male subjects. The injured leg of female subjects demonstrated more knee varus than that of the uninjured leg of female subjects. Regarding gender differences, female subjects demonstrated significantly more external hip rotation and knee valgus than male subjects did in both the injured and uninjured legs (P < 0.05). The current kinematic study exhibited biomechanical characteristics of female ACL-injured subjects compared with that of control groups. Kinematic correction during single leg half squat would reduce ACL reinjury in female ACL-injured subjects.

Roles of CLCA and CFTR in electrolyte re-absorption from rat saliva.

A molecular basis for Cl- re-absorption has not been well-characterized in salivary ductal cells. Previously, we found strong expression of a rat homologue proposed to be Ca2+-dependent Cl- channels (rCLCA) in the intralobular ducts of the rat submandibular gland. To address the question as to whether rCLCA and cystic fibrosis transmembrane conductance regulator (CFTR) are involved in Cl- re-absorption, we evaluated the electrolyte content of saliva from glands pre-treated with a small interfering RNA (siRNA). Retrograde injection into a given submandibular duct of an siRNA designed to knock down either rCLCA or CFTR reduced the expression of each of the proteins. rCLCA and CFTR siRNAs significantly increased Cl- concentration in the final saliva during pilocarpine stimulation. These results represent the first in vivo evidence for a physiological significance of rCLCA, along with CFTR, in transepithelial Cl- transport in the ductal system of the rat submandibular gland.

Acute monocytic leukaemia in a cat.

A three-year-old cat with lymphadenopathy, non-regenerative anaemia and marked leucocytosis (171.3 x 10(9) white blood cells/l) was diagnosed with monocytic leukaemia and treated with a combination of anticancer drugs. A number of mature and immature monocyte-like cells were detected in the peripheral blood and bone marrow; they proved to be monocytic cells by cytochemical examination and an analysis of their cell surface phenotype, indicating that the cat suffered from acute myeloid leukaemia, subclassified as monocytic leukaemia (M5). Treatment with cytarabine, doxorubicin, vincristine and prednisolone greatly reduced the number of blast cells in the cat's peripheral blood and bone marrow. The cat was in partial remission for 67 days and survived for 95 days after it was first examined.

Effects of long-term administration of methionine on vascular endothelium in rabbits.

BACKGROUND AND AIM: We studied the effects of long-term methionine administration on the vascular endothelium of Japanese white rabbits. METHODS AND RESULTS: Eleven rabbits were divided into a control group (n = 6) and a methionine-fed group (n = 5), and reared for 22 weeks. Blood samples were collected at baseline and after 22 weeks for the measurement of serum homocysteine and cysteine, serum lipids and serum superoxide dismutase activity. At the end of experiments, the animals were sacrificed, and the thoracic aorta was removed for the measurement of isometric tension and histopathological examination. The blood samples taken from the methionine group in the 22nd week showed slight but significant increases in serum homocysteine and cysteine levels (Hcy: 13.7 +/- 1.4 vs 21.0 +/- 4.9, p < 0.01; Cys: 241.6 +/- 37.8 vs 342.6 +/- 35.0, p < 0.01). In the isometric tension experiments, the methionine group had a significantly decreased (p < 0.01) vasodilatation reaction induced by acetylcholine, an endothelium-dependent vasodilator. The histopathological examination (immunostaining in response to eNOS and tissue factor) showed significant increases in endothelium expression in the methionine group before atherosclerotic changes appeared. CONCLUSIONS: The above results suggest that vascular endothelial dysfunction played an important role in the atherosclerosis occurring after excess methionine feeding.