Endoscopic Cytology for the Diagnosis of Chronic Enteritis and Intestinal Lymphoma in Dogs.

Although cytology is a rapid diagnostic procedure in dogs, the cytologic criteria of endoscopic biopsies for chronic enteritis and intestinal lymphoma are not well defined. An immediate diagnosis using cytology would benefit patients by enabling prompt initiation of therapy. The objective of this study was to investigate the correlation between the results of endoscopic cytology and histopathology. In this study, 167 dogs with clinical signs of chronic gastrointestinal disease were included. On the basis of histopathology, the following diagnoses were determined: lymphocytic-plasmacytic enteritis in 93 dogs; eosinophilic enteritis in 5 dogs; small cell intestinal lymphoma in 45 dogs; and large cell intestinal lymphoma in 24 dogs. Two clinical pathologists retrospectively evaluated the endoscopic cytology of squash-smear preparations. The cytologic diagnoses of inflammation, small cell lymphoma, and large cell lymphoma were based on the severity of lymphocyte infiltration, the size of infiltrated lymphocytes, and eosinophil/mast cell infiltration. The clinical severity score was significantly increased along with the degree of lymphocyte infiltration evaluated by cytology. The cytologic diagnosis was in complete agreement with the histopathologic diagnosis in 136 of 167 (81.4%) cases. For the differentiation between enteritis and lymphoma, endoscopic cytology had a sensitivity of 98.6%, a specificity of 73.5%, a positive predictive value of 72.3%, and a negative predictive value of 98.6%. The log-rank test and Cox regression analysis showed that the results of cytology predicted the prognosis. These results suggest that endoscopic cytology is a useful technique to aid diagnosis of intestinal inflammation and lymphoma in dogs.

Characterization of SPATA5-related encephalopathy in early childhood.

Mutations in SPATA5 have recently been shown to result in a phenotype of microcephaly, intellectual disability, seizures, and hearing loss in childhood. Our aim in this report is to delineate the SPATA5 syndrome as a clinical entity, including the facial appearance, neurophysiological, and neuroimaging findings. Using whole-exome sequencing and Sanger sequencing, we identified three children with SPATA5 mutations from two families. Two siblings carried compound heterozygous mutations, c.989_991del (p.Thr330del) and c.2130_2133del (p.Glu711Profs*21), and the third child had c.967T>A (p.Phe323Ile) and c.2146G>C (p.Ala716Pro) mutations. The three patients manifested microcephaly, psychomotor retardation, hypotonus or hypertonus, and bilateral hearing loss from early infancy. Common facies were a depressed nasal bridge/ridge, broad eyebrows, and retrognathia. Epileptic spasms or tonic seizures emerged at 6-12 months of age. Interictal electroencephalography showed multifocal spikes and bursts of asynchronous diffuse spike-wave complexes. Augmented amplitudes of visually evoked potentials were detected in two patients. Magnetic resonance imaging revealed hypomyelination, thin corpus callosum, and progressive cerebral atrophy. Blood copper levels were also elevated or close to the upper normal levels in these children. Clinical delineation of the SPATA5-related encephalopathy should improve diagnosis, facilitating further clinical and molecular investigation.

Changes in Foxp3-Positive Regulatory T Cell Number in the Intestine of Dogs With Idiopathic Inflammatory Bowel Disease and Intestinal Lymphoma.

Although regulatory T cells (Tregs) play an integral role in immunologic tolerance and the maintenance of intestinal homeostasis, their involvement in canine gastrointestinal diseases, including idiopathic inflammatory bowel disease (IBD) and intestinal lymphoma, remains unclear. Here we show altered numbers of forkhead box P3 (Foxp3)-positive Tregs in the intestine of dogs with IBD and intestinal lymphoma. IBD was diagnosed in 48 dogs; small cell intestinal lymphoma was diagnosed in 46 dogs; large cell intestinal lymphoma was diagnosed in 30 dogs; and 25 healthy beagles were used as normal controls. Foxp3-positive Tregs in the duodenal mucosa were examined by immunohistochemistry and immunofluorescence. Duodenal expression of interleukin-10 mRNA was quantified by real-time reverse transcription polymerase chain reaction. The number of Foxp3-positive lamina propria cells and the expression of interleukin-10 mRNA were significantly lower in dogs with IBD than in healthy dogs and dogs with intestinal lymphoma. The number of Foxp3-positive intraepithelial cells was higher in dogs with small cell intestinal lymphoma. Some large cell intestinal lymphoma cases had high numbers of Foxp3-positive cells, but the increase was not statistically significant. Double-labeling immunofluorescence showed that CD3-positive granzyme B-negative helper T cells expressed Foxp3. In small cell intestinal lymphoma cases, the overall survival of dogs with a high Treg density was significantly worse than that of dogs with a normal Treg density. These results suggest that a change in the number of Foxp3-positive Tregs contributes to the pathogenesis of canine IBD and intestinal lymphoma by disrupting mucosal tolerance and suppressing antitumor immunity, respectively.

Pathologic Features of Colorectal Inflammatory Polyps in Miniature Dachshunds.

The histopathologic characteristics of colorectal inflammatory polyps that formed in Miniature Dachshunds were compared with those of other colorectal proliferative lesions, including adenomas and adenocarcinomas. Fifty-three colorectal polypoid lesions were histopathologically classified into inflammatory polyps (26 cases), adenoma (18 cases), and adenocarcinoma (9 cases). All 26 dogs that were diagnosed with inflammatory polyps were Miniature Dachshunds, indicating that colorectal inflammatory polyps exhibit a marked predilection for this breed. The inflammatory polyps had complex histopathologic features and were classified into 3 stages based on their epithelial composition. In early stage (stage 1), the polyps tended to exhibit a thickened mucosa containing hyperplastic goblet cells, dilated crypts filled with a large amount of mucus, and mild lymphocyte and macrophage infiltration. In later stages (stages 2 and 3), more severe neutrophil infiltration, interstitial mucus accumulation, granulation tissue, and occasional osteoid tissue were seen. Also, a few small foci of dysplastic epithelial cells were detected. The hyperplastic goblet cells, which were a major component of the epithelium of the inflammatory polyps, were positive for cytokeratin 20 (CK20), while the dysplastic epithelial cells found in inflammatory polyps (stage 3) and the tumor cells of the adenomas and adenocarcinomas were negative for CK20. These CK20-negative epithelial cells exhibited cytoplasmic and nuclear immunoreactivity for beta-catenin. In addition, the epithelial cells in the inflammatory polyps demonstrated significantly higher cyclooxygenase 2 and fibroblast growth factor 2 expression than did those of the adenomas and adenocarcinomas, suggesting that the arachidonate cascade is involved in the development of colorectal inflammatory polyps in miniature dachshunds.

Decoherence of Near-Surface Nitrogen-Vacancy Centers Due to Electric Field Noise.

We show that electric field noise from surface charge fluctuations can be a significant source of spin decoherence for near-surface nitrogen-vacancy (NV) centers in diamond. This conclusion is based on the increase in spin coherence observed when the diamond surface is covered with high-dielectric-constant liquids, such as glycerol. Double-resonance experiments show that improved coherence occurs even though the coupling to nearby electron spins is unchanged when the liquid is applied. Multipulse spin-echo experiments reveal the effect of glycerol on the spectrum of NV frequency noise.

Demonstration of the cell clonality in canine hematopoietic tumors by X-chromosome inactivation pattern analysis.

X-chromosome inactivation pattern (XCIP) analysis has been widely used to assess cell clonality in various types of human neoplasms. In this study, a polymerase chain reaction-based canine XCIP analysis of the androgen receptor (AR) gene was applied for the assessment of cell clonality in canine hematopoietic tumors. This XCIP analysis is based on the polymorphic CAG repeats in the AR gene and the difference of methylation status between active and inactive X chromosomes. We first examined the polymorphisms of 2 CAG tandem repeats in the AR gene in 52 male and 150 female dogs of various breeds. The 2 polymorphic CAG repeats contained 9 to 12 and 10 to 14 CAGs in the first and second CAG repeats, respectively. Of the 150 female dogs, 74 (49.3%) were heterozygous for the first and/or second polymorphic CAG tandem repeats, indicating the utility of XCIP analysis in these dogs. Canine XCIP analysis was then applied to clinical samples from female dogs with canine high-grade lymphoma, chronic myelogenous leukemia, acute myelogenous leukemia, and benign lymph node hyperplasia. Of 10 lymphoma cell samples, 9 (90%) showed skewed XCIPs, indicating their clonal origins, whereas all the nonneoplastic lymph node samples showed balanced XCIPs. Moreover, bone marrow specimen from a dog with acute myelogenous leukemia and peripheral leukocyte specimens from 2 dogs with chronic myelogenous leukemia showed skewed XCIPs. XCIP analysis was successfully employed to demonstrate the cell clonality of canine hematopoietic tumors in this study and will be applicable to evaluate the clonality in various proliferative disorders in dogs.

Probing surface noise with depth-calibrated spins in diamond.

Sensitive nanoscale magnetic resonance imaging of target spins using nitrogen-vacancy (NV) centers in diamond requires a quantitative understanding of dominant noise at the surface. We probe this noise by applying dynamical decoupling to shallow NVs at calibrated depths. Results support a model of NV dephasing by a surface bath of electronic spins having a correlation rate of 200 kHz, much faster than that of the bulk N spin bath. Our method of combining nitrogen delta-doping growth and nanoscale depth imaging paves a way for studying spin noise present in diverse material surfaces.

Multipulse double-quantum magnetometry with near-surface nitrogen-vacancy centers.

We discuss multipulse magnetometry that exploits all three magnetic sublevels of the S=1 nitrogen-vacancy center in diamond to achieve enhanced magnetic field sensitivity. Based on dual frequency microwave pulsing, the scheme is twice as sensitive to ac magnetic fields as conventional two-level magnetometry. We derive the spin evolution operator for dual frequency microwave excitation and show its effectiveness for double-quantum state swaps. Using multipulse sequences of up to 128 pulses under optimized conditions, we show enhancement of the SNR by up to a factor of 2 in detecting NMR statistical signals, with a 4× enhancement theoretically possible.

Multilayer coated grazing incidence condenser for large numerical aperture objective at wavelength of 4.5 nm.

A grazing incidence condenser is developed for objectives with large numerical aperture working in Carbon-window wavelength region (λ=4.4-5.0 nm) with the use of a point light source. The condenser is composed of four pieces of toroidal mirrors and a piece of the mirror was fabricated to evaluate the performance of the mirror. The radii of the toroidal mirror are determined by ray-trace calculation, and each radius of the mirror substrate and the roughness of the polished surface were evaluated to satisfy the designed parameter. A Co/C reflection multilayer is also designed to reflect soft x-ray light at 4.5 nm wavelength, and the reflection multilayer was deposited on the mirror surface. Measured reflectance of the toroidal mirror with the reflection multilayer is higher than 0.32 at 4.5 nm wavelength.

Modulation of pain perception through transcranial alternating current stimulation and its nonlinear relationship with the simulated electric field magnitude.

BACKGROUND: Oscillatory activities observed in multiple regions are closely associated with the experience of pain. Specifically, oscillatory activities within the theta- and beta-frequency bands, observed in the left dorsolateral prefrontal cortex (DLPFC), have been implicated in pain perception among healthy individuals and those with chronic pain. However, their physiological significance remains unclear. METHODS: We explored the modulation of pain perception in healthy individuals by theta- and beta-band transcranial alternating current stimulation (tACS) over the left DLPFC and examined the relationship between the modulation effect and magnitude of the electric field elicited by tACS in the left DLPFC using computational simulation. RESULTS: Our findings revealed that both theta- and beta-tACS increased the heat pain threshold during and after stimulation. Notably, the simulated electric field magnitude in the left DLPFC exhibited an inverted U-shaped relationship with the pain modulation effect for theta-tACS. CONCLUSIONS: Our study findings suggested that there would be an optimal electric field strength to produce a high analgesic effect for theta-tACS. SIGNIFICANCE: The application of theta- and beta-tACS interventions targeting the left DLPFC might facilitate the treatment of chronic pain. Furthermore, the attainment of effective pain modulation via theta-tACS over the DLPFC warrants the use of optimal stimulus intensity.

Pharmacokinetics of micafungin in patients undergoing allogeneic hematopoietic stem cell transplantation.

OBJECTIVES: Micafungin (MCFG) is an antifungal agent that is widely used for the treatment of invasive fungal infection. Although the pharmacokinetics of MCFG is considered to depend on the hepatic metabolism, the impact of hepatic function on the pharmacokinetics of MCFG has been inconsistent among previous studies. The object of this study was to evaluate the relationship between plasma MCFG concentration and clinical and laboratory data. PATIENTS AND METHODS: We examined the plasma concentration of MCFG in 10 patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT). MCFG at 150 mg/day was administered intravenously a median of 58.5 days after HSCT. Trough and peak concentrations of MCFG (Cmin and Cmax) were measured at a median of 5.5 days after the first administration of MCFG. RESULTS: The presence of graft-versus-host disease involving the liver at blood sampling was associated with significantly higher Cmin and Cmax of MCFG. Among the laboratory data, Cmin and Cmax were significantly higher in patients with severely impaired hepatic function defined as serum total bilirubin (TBi) level >5 mg/dL and/or serum gamma-glutamyltransferase (γ-GTP) level >500 IU/L, but the presence of mildly impaired hepatic function defined as serum TBi level >2 mg/dL and/or serum γ-GTP level >200 IU/L did not affect Cmin and Cmax. Renal function did not show significant impact on Cmin and Cmax. CONCLUSION: These findings suggest that the pharmacokinetics of MCFG is affected only by severely impaired liver function.

X-chromosome inactivation pattern analysis for the assessment of cell clonality in cats.

X-chromosome inactivation pattern (XCIP) analysis has been widely used to assess cell clonality in various types of neoplasms in humans. In the present study, a polymerase chain reaction-based feline XCIP analysis using the feline androgen receptor gene was developed. To construct the system of the analysis, polymorphism in CAG tandem repeats within the feline androgen receptor gene was explored using somatic DNAs from 50 male and 103 female cats. CAG tandem repeats in exon 1 of the feline androgen receptor gene were found to be polymorphic, containing 15 to 22 CAG repeats. Of the 103 female cats, 70 (68%) were heterozygous for the number of CAG repeats, indicating the possible usefulness of XCIP analysis in cats. Application of the feline XCIP analysis to 3 feline mammary gland adenocarcinoma cell lines revealed distinctly skewed XCIPs in these cell lines, indicating their clonal origins. Twelve (80%) of the 15 primary tissue/cell samples obtained from cats with various neoplastic diseases showed skewed XCIPs. Moreover, bone marrow samples from 3 cats with myelodysplastic syndrome were also found to have skewed XCIPs. The polymerase chain reaction-based XCIP analysis developed in this study can provide information on cell clonality in female cats, potentially facilitating the differential diagnosis of various disorders in cats.

Biological effect of tyrosine kinase inhibitors on three canine mast cell tumor cell lines with various KIT statuses.

Tyrosine kinase inhibitors (TKIs) can be important in the treatment of canine mast cell tumor (cMCT). Meanwhile, some TKIs have been identified as substrates for ABCB1. The inhibitory effect of four TKIs (axitinib, imatinib, masitinib, and vatalanib) for proliferation and phosphorylation of c-Kit receptor as well as the expression and function of ABCB1 were investigated in three cMCT cell lines (HRMC, VIMC1, and CMMC1). The IC(50) values of the TKIs in HRMC, the only cell line with wild-type KIT, were clearly higher than those in CMMC1 and VIMC1. In HRMC and CMMC1, both the growth and phosphorylation of c-Kit receptor were suppressed proportionally by the TKIs. VIMC1 required higher concentrations for the inhibition of c-Kit receptor phosphorylation than those in cell growth. The treatment with cyclosporine increased the effects of the TKIs on VIMC1 since ABCB1 was expressed in VIMC1. The results indicated that cMCT cell lines harboring wild-type KIT had lower sensitivity to TKIs. The growth of VIMC1 was seemingly reduced by TKIs through the inhibition of other tyrosine kinases than c-Kit receptor. There was little influence of ABCB1 on TKI effects to the proliferation of VIMC1. These results will be helpful to understand the different sensitivity to TKIs in cMCT patients.

Expression of fibrosis-related genes in canine chronic hepatitis.

Molecular regulation of fibrosis in chronic canine hepatitis is poorly understood. The authors employed quantitative polymerase chain reaction (PCR) to determine the expression levels of genes reported to be related to fibrosis in other species (human, mouse, and rat) and to elucidate the relationship of these genes with the degree of fibrosis and the presence or absence of ascites and/or jaundice in dogs with hepatitis. Nine fibrosis-related genes were assayed: PDGFB, PDGFD, MMP2, TIMP1, THBS1, COL1A1, COL3A1, TGFB1, and TGFB2. Liver samples of 15 dogs with chronic hepatitis and 4 healthy control dogs were obtained via laparoscopic biopsy and subjected to histologic and quantitative PCR analyses. The expression of all 9 genes showed significant positive correlation (P<.01, r>.70) with the degree of fibrosis. Furthermore, the expression levels of all genes except TGFB1 were significantly higher (P<.05) in dogs with hepatic failure-related symptoms (ascites/jaundice). Results suggest that these 9 genes are integral to the development of fibrosis in canine chronic hepatitis.

Construction and validation of a scoring system to predict resistance to chemotherapeutic drugs using gene expression profiles in canine lymphoma.

The present study aimed to verify the changes in the expression levels of 13 candidate genes associated with chemotherapy resistance and to construct a scoring system to predict resistance to these drugs. The expression levels of the 13 candidate genes were compared between 20 dogs with lymphoma that were sensitive to drugs used in CHOP-based protocol and 16 dogs with lymphoma that were resistant to these drugs. The expression levels of six genes; ASNS, CCR3, CALCA, FCER1A, LOC448801, and EDNRB were significantly different between the two groups. A scoring system to predict resistance to cyclophosphamide, doxorubicin and vincristine, which are used in CHOP-based protocol, was constructed based on expression levels of the six genes in these 36 dogs using logistic regression models. After internal validation, sensitivity and specificity of the scoring system were 0.759 and 0.853, respectively. External validation was conducted in another cohort of 33 dogs with lymphoma, and sensitivity and specificity of the scoring system were 0.800 and 0.696, respectively. In conclusion, this study identified six genes associated with resistance to drugs used in CHOP-based protocol in canine lymphoma and proposed a novel scoring system to predict resistance to these drugs. This system might be beneficial in selecting the most appropriate chemotherapy protocol for individual dogs with lymphoma.

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.

Evaluation of norovirus removal performance in a coagulation-ceramic microfiltration process by using recombinant norovirus virus-like particles.

Norovirus (NV) is a prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis worldwide. Because of the lack of a cell culture system or an animal model for this virus, studies on drinking water treatment such as separation and disinfection processes are still hampered. In the present study, we investigated NV removal performance as particles during a coagulation-ceramic microfiltration (MF) process by using recombinant NV virus-like particles (rNV-VLPs), which are morphologically and antigenically similar to native NV. We also experimentally investigated the behaviors of two widely accepted surrogates for pathogenic waterborne viruses, bacteriophages Qbeta and MS2, for comparison with the behavior of rNV-VLPs. More than 4-log removal was observed for rNV-VLPs with a 1.08 mg-Al/L dose of polyaluminium chloride in the coagulation-ceramic MF process. This high removal ratio of rNV-VLPs satisfies the U.S. Environmental Protection Agency requirement of 4-log removal or inactivation. In addition, the removal ratios of Qbeta and MS2 were approximately 2-log and 1-log, smaller than the ratio of rNV-VLPs. Accordingly, both bacteriophages have the potential to become appropriate surrogates for native NV in the coagulation-ceramic MF process, and, of the two, Qbeta is the more conservative surrogate.

Geosmin and 2-methylisoborneol adsorption on super-powdered activated carbon in the presence of natural organic matter.

Geosmin and 2-methylisoborneol (2-MIB) are naturally occurring compounds responsible for musty-earthy-odors in surface water supplies. They are a severe problem confronting utilities worldwide. Adsorption by powdered activated carbon (PAC) is a widely used process to control this problem, but it has low efficiency, which engenders large budget spending for utilities services. Super-powdered activated carbon (S-PAC) is activated carbon with much finer particles than those of PAC. Experiments on geosmin and 2-MIB adsorptions on S-PAC and PAC were conducted. Geosmin and 2-MIB adsorption capacities on S-PAC were not smaller than those on PAC although natural organic matter, which adversely impacted the adsorption capacity of geosmin and 2-MIB, was more adsorbed on S-PAC than on PAC, meaning that the adsorption competition is less severe for S-PAC than for PAC.

Clear evidence for element partitioning effects in a Ti-6Al-4V alloy by the first-principles phase field method.

Ti-6 wt% Al-4 wt% V (Ti64) is an α + ß titanium alloy, in which the alloying components strongly affect the mechanical properties. In this report, element partitioning effects in Ti64 are investigated by using the first-principles phase field (FPPF) method, which has recently been proposed by our group. In the FPPF method, the local free energy is calculated using a cluster expansion method in combination with density functional theory and the temperature effect is incorporated using potential renormalization theory. We have succeeded in identifying enrichment of Al (V) in the α (ß) phase, i.e., the clear evidence for the element partitioning effects of Al and V, without using any thermodynamical parameter. The transformation of the ß phase and the α phase in microstructure is investigated by varying the V and Al concentrations by a small amount. Our results are in excellent agreement with the recent experimental results, showing the validity of the FPPF method for ternary alloys.

Entry of opioid peptides into the central nervous system.

Cerebrovascular permeability of four modified opioid peptides--[D-Ala2]methionine enkephalin amide, beta-[D-Ala62,14C-Homoarg69]lipotropin 61 -69, alpha-[D-Ala2,14C-Homoarg9]endorphin, and beta-[D-Ala2,14C-Homoarg]endorphin--ranged from 1.4 to 3.9 X 10(-6) centimeters per second in brain regions of the conscous rat. These significant permeabilities should allow the peptides to fill the extracellular brain space with a half time of 3 to 11 minutes, as a result of a step increase in plasma concentration of unbound peptide.