Effect of dasatinib in a xenograft mouse model of canine histiocytic sarcoma and in vitro expression status of its potential target EPHA2.

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic tumor. Previously, the kinase inhibitor dasatinib was shown to have potent growth inhibitory activity against HS cells in vitro, possibly via targeting the EPHA2 receptor. Here, the in vivo effect of dasatinib in HS cells was investigated using a xenograft mouse model. Moreover, the expression status of EPHA2 was examined in six HS cell lines, ranging from insensitive to highly sensitive to dasatinib. In the HS xenograft mouse model, dasatinib significantly suppressed tumor growth, as illustrated by a decrease in mitotic and Ki67 indices and an increase in apoptotic index in tumor tissues. On Western blot analysis, EPHA2 was only weakly detected in all HS cell lines, regardless of sensitivity to dasatinib. Dasatinib likely results in the inhibition of xenograft tumor growth via a mechanism other than targeting EPHA2. The findings of this study suggest that dasatinib is a targeted therapy drug worthy of further exploration for the treatment of canine HS.

Selective growth inhibition by suppression of F1Fo ATPase in canine malignant melanoma cell lines.

Canine malignant melanoma (CMM) is a highly aggressive and fatal neoplasm. To identify potential therapeutic compounds and/or targets, 320 compounds were screened for their growth inhibitory activity in a CMM line (CMM-1) using a chemical library known to target specific signaling pathways/cell growth-related molecules. Among the compounds screened, the F1Fo ATPase inhibitor oligomycin showed potent growth inhibitory effects in CMM-1 cells, while exhibiting less toxic effects in a non-neoplastic control cell line (MDCK cells). The growth inhibitory effect of oligomycin A was then examined using six CMM lines and MDCK cells. Three CMM lines were highly sensitive to oligomycin A, with around 3000-20 000 times lower IC50 compared with oligomycin A-resistant CMM lines and MDCK cells. Oligomycin A-sensitive CMM-1 cells exhibited much greater oligomycin A-induced decreases in cellular ATP compared to oligomycin A-resistant cell lines. Although the oligomycins are clinically unsuitable because of its in vivo toxicity, these findings implicate the potential of F1Fo ATPase as a therapeutic target in a subset of CMM.

The detection of toxigenic Corynebacterium ulcerans from cats with nasal inflammation in Japan.

Corynebacterium ulcerans (toxigenic C. ulcerans) produces the diphtheria toxin, which causes pharyngeal and cutaneous diphtheria-like disease in people, and this bacterium is commonly detected in dogs and cats that are reared at home. It is considered dangerous when a carrier animal becomes the source of infection in people. To investigate the carrier situation of toxigenic C. ulcerans of cats bred in Japan, bacteria were isolated from 37 cats with a primary complaint of rhinitis in 16 veterinary hospitals in Osaka. Toxigenic C. ulcerans was detected in two of the cats. By drug sensitivity testing, the detected bacterium was sensitive to all investigated drugs, except clindamycin. It appears necessary to create awareness regarding toxigenic C. ulcerans infection in pet owners because this bacterium is believed to be the causative organism for rhinitis in cats.

Lactobacillus kunkeei YB38 from honeybee products enhances IgA production in healthy adults.

AIMS: To identify lactic acid bacterial isolates, which promote immunoglobulin A (IgA) production in honeybee products and honeybees (Apis mellifera). METHODS AND RESULTS: Pyrosequencing analysis of the microbiota of honeybee products and honeybees revealed the predominance of Lactobacillus kunkeei in honey, bee pollen, bee bread and royal jelly. Lactobacillus kunkeei was isolated from bee pollen, bee bread and honey stomach, and its effect on IgA production was evaluated in vitro. Heat-killed YB38 and YB83 isolates from bee pollen promoted IgA production in mouse Peyer's Patch cells and had little mitogenic activity or effect on IL-2 production in mouse spleen cells in comparison with Listeria monocytogenes, which does exhibit mitogen activity. A pilot study in 11 healthy adults showed that 4-week intake of 1000 mg day(-1) heat-killed YB38 increased secretory IgA (SIgA) concentrations and secretion in saliva with no adverse effects. CONCLUSION: Heat-killed Lact. kunkeei YB38 from bee pollen increases IgA production and may safely improve immune responsiveness. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of microbiota analysis of royal jelly and the immune efficacy of Lact. kunkeei from honeybee products in humans.

Timing of adjunctive therapy in the treatment of depression: a chart review.

INTRODUCTION: The objective of this study was to examine the evolution of antidepressant switch and adjunctive therapy. METHODS: This chart review was conducted at 6 primary psychiatric clinics or hospitals, in Tokyo, Japan. A chart review of longitudinal prescriptions was conducted regarding 633 outpatients with major depressive disorder for up to 2 years after their first visit. Patients who had already received antidepressants prior to the visit were excluded. RESULTS: 22.6% (N=143) of the patients completed or continued the outpatient treatment over the 2 years while 27 (4.3%), 23 (3.6%), and 439 (69.4%) patients discontinued it due to hospitalization, referral to another clinic, and loss to follow-up, respectively. A total of 597 episodes of antidepressant treatment were identified. Among them, 482 episodes (80.7%) were associated with the suggested dose ranges while antidepressant drugs were under-dosed in 19.3% (N=115) of the episodes. 50 patients (7.9%) received adjunctive therapy; it was employed after a median of only one antidepressant had been tried. CONCLUSION: Psychiatrists may be hasty in prescribing an adjunctive therapy in the treatment of depression.

Glutamate biosensor imaging reveals dysregulation of glutamatergic pathways in a model of developmental cortical malformation.

Cortical malformations can cause intractable epilepsy, but the underlying epileptogenic mechanisms are poorly understood. We used high-speed glutamate biosensor imaging to ask how glutamatergic signaling is altered in cortical malformations induced by neonatal freeze-lesions (FL). In non-lesion neocortical slices from 2 to 8week old rats, evoked glutamate signals were symmetrical in the medio-lateral axis and monotonic, correlating with simple, brief (≈50ms) local field potentials (LFPs). By contrast, in FL cortex glutamate signals were prolonged, increased in amplitude, and polyphasic, which paralleled a prolongation of the LFP. Using glutamate biosensor imaging, we found that glutamate signals propagated throughout large areas of FL cortex and were asymmetric (skewed toward the lesion). Laminar analysis demonstrated a shift in the region of maximal glutamate release toward superficial layers in FL cortex. The ability to remove exogenous glutamate was increased within the FL itself but was decreased in immediately adjacent regions. There were corresponding alterations in astrocyte density, with an increase within the lesion and a decrease in deep cortical layers surrounding the lesion. These findings demonstrate both network connectivity and glutamate metabolism are altered in this cortical malformation model and suggests that the regional ability of astrocytes to remove released glutamate may be inversely related to local excitability.

Surgical repair of an unroofed coronary sinus in a dog.

A 5-year-old, 7.9 kg castrated male Miniature Dachsund presented with heart enlargement on radiography. The dog was asymptomatic. Echocardiography revealed a tubular structure running along the posterior wall of the left atrium and connecting to the right atrium on the caudal side of the left atrium and annulus, which was presumed to be a dilated coronary sinus. After confirming a shunt between the left atrium and coronary sinus by cardiovascular catheterization, an unroofed coronary sinus was diagnosed. Open-heart surgery using cardiopulmonary bypass was performed through left atriotomy. The defect between the left atrium and the coronary sinus was closed by suturing. The cardiac enlargement improved after surgery. The dog was still alive 1227 days after surgery without clinical signs.

Predictive values of immune indicators on respiratory failure in the early phase of COVID-19 due to Delta and precedent variants.

Background: Immune response indicators in the early phase of COVID-19, including interferon and neutralizing responses against SARS-CoV-2, which predict hypoxemia remains unclear. Methods: This prospective observational study recruited patients hospitalized with COVID-19 (before emergence of omicron variant). As the immune indicators, we assessed the serum levels of IFN-I/III, IL-6, CXCL10 and VEGF, using an ELISA at within 5 days after the onset of symptoms, and serum neutralizing responses using a pseudovirus assay. We also assessed SARS-CoV-2 viral load by qPCR using nasal-swab specimens and serum, to assess the association of indicators and viral distribution. Results: The study enrolled 117 patients with COVID-19, of which 28 patients developed hypoxemia. None received vaccine before admission. Serum IFN-I levels (IFN-α and IFN-ß), IL-6, CXCL10, LDH and CRP were significantly higher in patients who developed hypoxemia. A significant association with nasopharyngeal viral load was observed only for IFN-I. The serum levels of IFN-α, IL-6, CXCL10 were significantly associated with the presence of RNAemia. Multivariable analysis showed higher odds ratio of IFN-α, with cut-off value of 107 pg/ml, in regard to hypoxemia (Odds ratio [OR]=17.5; 95% confidence interval [CI], 4.7-85; p<0.001), compared to those of IL-6, >17.9 pg/ml (OR=10.5; 95% CI, 2.9-46; p<0.001). Conclusions: This study demonstrated that serum IFN-α levels in the early phase of SARS-CoV-2 infection strongly predict hypoxemic respiratory failure in a manner different from that of the other indicators including IL-6 or humoral immune response, and instead sensitively reflect innate immune response against SARS-CoV-2 invasion.

Morphological changes and viability of Cryptosporidium parvum sporozoites after excystation in cell-free culture media.

Cryptosporidium parvum, belonging to the phylum Apicomplexa, is a major cause of waterborne gastroenteritis throughout the world. The sporozoites are thought to invade host enterocytes using an active process termed gliding motility. However, the biological and morphological changes within the sporozoites during this process are not fully understood. In the present study, excysted sporozoites of C. parvum were analysed ultrastructurally in vitro and their viability was evaluated using fluorescent dyes. The sporozoites excysted from oocysts changed morphologically from banana-shaped to rod-shaped and finally to a rounded shape, in culture media in 3 h. Transmission microscopy revealed that the distance between the apical end and the nucleus was markedly reduced, dense granules were present close to the rhoptry in the apical region, amylopectin granules were absent, and membranes of round sporozoites were less clear. A fluorescent assay showed that the rate of survival decreased from 89% to 56% at 0-3 h (84.3% for banana-shaped and 49.2% for rod-shaped sporozoites). Therefore, post-excysted sporozoites in vitro underwent morphological changes and a rapid loss of viability. This staining method is useful, inexpensive and provides an alternative to more costly and intensive flow cytometric assays or infectivity assays with host cells in vitro.

Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin- mediated cell-cell adhesion.

The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target of Cdc42 and Rac1, was localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts expressing E-cadherin (EL cells), and interacted with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induced the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, resulted in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway.

Electron microscopic observation of cytoskeletal frame structures and detection of tubulin on the apical region of Cryptosporidium parvum sporozoites.

Cryptosporidium parvum is an intracellular protozoan parasite belonging to the phylum Apicomplexa, and a major cause of waterborne gastroenteritis throughout the world. Invasive zoites of apicomplexan parasites, including C. parvum, are thought to have characteristic organelles on the apical apex; however, compared with other parasites, the cytoskeletal ultrastructure of C. parvum zoites is poorly understood. Thus, in the present study, we ultrastructurally examined C. parvum sporozoites using electron microscopy to clarify the framework of invasive stages. Consequently, at the apical end of sporozoites, 3 apical rings and an electron-dense collar were seen. Two thick central microtubules were seen further inside sporozoites and extended to the posterior region. Using anti-alpha and -beta tubulin antibodies generated from sea urchin and rat brain, both antibodies cross-reacted at the apical region of sporozoites in immunofluorescent morphology. The molecular mass of C. parvum alpha tubulin antigen was 50 kDa by Western blotting and the observed apical cytoskeletal structures were shown to be composed of alpha tubulin by immunoelectron microscopy. These results suggested that C. parvum sporozoites were clearly different in their cytoskeletal structure from those of other apicomplexan parasites.

Canine squamous cell carcinoma cell lines with high expression of survivin are sensitive to survivin inhibitor YM155.

Treatment of unresectable canine squamous cell carcinoma (SCC) remains challenging and new therapeutic strategies are needed. Survivin is a member of the inhibitor of apoptosis protein family and its inhibitor, YM155, is a potential anti-tumour agent. In the present study, 10 canine tumour cell lines (representing eight different tumour types) were screened for sensitivity to YM155; the drug potently inhibited the growth of the HAPPY SCC cell line. The growth inhibitory properties of YM155 were then examined in more detail using a panel of seven SCC cell lines. YM155 inhibited the growth of the cell lines HAPPY and SQ4; in contrast to the other lines in the panel, these two cell lines had high levels of expression of survivin. In HAPPY cells, YM155 inhibited expression of the survivin gene at the transcriptional level. In contrast, YM155 down-regulated survivin at the post-transcriptional level in SQ4 cells. YM155 suppressed cell growth in HAPPY cells, mostly via induction of apoptosis, but this was not the case in SQ4 cells. Two canine SCC cell lines with high cellular expression of survivin were sensitive to YM155. The possible underlying mechanisms of the cytotoxic effect of YM155 in these cell lines were different. One cell line had down-regulation of survivin mRNA and protein expression, associated with induction of apoptotic cell death. The other cell line had post-transcriptional down-regulation of survivin expression and subsequent induction of non-apoptotic cell death. Targeting survivin with YM155 is a potential approach for the treatment of canine SCCs with high expression of survivin.

Survey of Japanese layer farms for Salmonella enteritidis with vaccination- and infection-specific antigens for egg yolk antibodies.

Japanese layer farms were surveyed for Salmonella Enteritidis vaccination and infection with specific antigens for egg yolk antibodies with the use of vaccination-specific antigen Salmonella Enteritidis FliC-specific 9-kDa polypeptide (SEP9) and infection-specific antigen deflagellated Salmonella Enteritidis whole cell (DEWC). The specific antibodies in eggs from 201 commercial layer farms throughout Japan were surveyed. The percentages of farm flocks with a mean enzyme-linked immunosorbent assay (ELISA) titer of over 0.1 were 56.2% (113 of 201) in DEWC-ELISA and 22.3% (45 of 201) in SEP9-ELISA. Flocks indicating high titers in SEP9-ELISA always showed high titers in DEWC-ELISA. Because both specific antibody titers of the vaccinated flocks monitored long term remained high throughout life, flocks with high titers of both ELISAs in this survey must be vaccinated. On the other hand, 34.3% (69 of 201) of flocks had high titers of DEWC-specific antibody alone. Because Salmonella Enteritidis infection induces the DEWC-specific antibody but not the SEP9-specific antibody, detecting only high ELISA titers of DEWC-specific antibody can be an effective monitoring tool for Salmonella Enteritidis exposure rather than vaccination. These results suggest that vaccination programs in Japanese layer farms would be insufficient to control Salmonella Enteritidis infection, and egg screening to detect specific antibodies would be valuable in obtaining the necessary information to control Salmonella Enteritidis infection.

Effect of adenosine deaminase inhibitors on myocardial reactive hyperaemia following brief coronary occlusions.

Effect of two agents of adenosine deaminase inhibitor, 8-azaguanine and adenine, on myocardial reactive hyperaemia was tested in the anaesthetised open-chest dog. Reactive hyperaemic flow response of the circumflex coronary artery was observed following 5, 10, 15, 20 and 30 s coronary occlusions before, during and after infusion of 8-azaguanine and adenine, which are known as adenosine deaminase inhibitors. Intracoronary infusion of 8-azaguanine and adenine caused the minimum increase in the baseline coronary flow. Both the nucleic acids shifted the dose response curve of adenosine to the left. 8-azaguanine enhanced volume response of flow at all occlusion intervals tested. The infused dose of adenine also intensified volume response of flow after 5, 15, 20 and 30 s occlusions. Fifteen minutes after termination of the nucleic acid infusions, the reactive hyperaemia returned towards control levels. The results suggest that 8-azaguanine and adenine enhance myocardial reactive hyperaemia possibly by inhibiting adenosine deaminase to degradate myocardial interstitial adenosine to inosine.

Oxygen metabolism of the hypertrophic right ventricle in open chest dogs.

STUDY OBJECTIVE: The aim was to investigate oxygen metabolism of the hypertrophic right ventricle in anaesthetised open chest dogs. DESIGN: Right ventricular hypertrophy was induced by right ventricular pressure overload with banding the pulmonary artery for six months. Coronary blood flow and myocardial oxygen metabolism of the hypertrophic right ventricle were determined during control and after increasing right ventricular oxygen consumption, and compared with those of the normal right and left ventricles. SUBJECTS: Seven mongrel dogs with right ventricular hypertrophy and 21 normal dogs were used. All were anaesthetised with pentobarbitone sodium. MEASUREMENTS AND MAIN RESULTS: Oxygen extraction [(A-V)O2] of the hypertrophic right ventricular myocardium was greater than that of normal right ventricle in controls and almost identical to the (A-V)O2 of the normal left ventricle. It showed no increase when coronary blood flow and right ventricular oxygen consumption were raised in response to a further elevation of the right ventricular pressure and isoprenaline infusion. However, the right ventricular interventions which increased right ventricular oxygen consumption produced an elevation of (A-V)O2 of the right ventricle with an increase in right coronary blood flow. CONCLUSIONS: Higher oxygen extraction during control and no response of oxygen extraction of the hypertrophied right ventricle in response to stimuli which increase right ventricular oxygen consumption indicate that oxygen supply to the hypertrophic right ventricle is different from that of the normal right ventricle, and is more like that of the left ventricle.

Identification of novel tumour-associated antigens in canine mammary gland tumour.

UNLABELLED: Canine mammary gland tumour (MGT) is the most common neoplasm in female dogs and has similar biological characteristics to human MGT. Spontaneous canine MGT is a more attractive clinical model in oncological research than that of the murine experimental model. Tumour-associated antigens (TAAs), which are produced in tumour cells, are applied as tumour markers, tumour vaccine antigens and molecular targets of therapeutic drugs. In this study, we have primarily identified 13 different TAAs of canine MGT by serological immunoscreening of cDNA expression library. The results of serological mini-arrays of identified antigens showed that CCDC41 antigen specially reacted with 35% of sera from MGT-dogs and did not react with control sera. We also found that HSPH1 mRNA expression levels increased significantly in MGT tissues. These findings will contribute to the development of diagnostic technologies and translational target therapies for dogs. CLINICAL RELEVANCE: HSPH1, which is strongly expressed in the tumour tissue, will be a possible vaccine antigen of canine MGT.

Effects of octreotide, a somatostatin analogue, on gastric function evaluated by real-time ultrasonography.

BACKGROUND: Somatostatin exerts inhibitory effects on physiological functions in the gastrointestinal tract. The actions differ, however, depending on the test meal, dose, and other factors. AIMS: To determine by use of ultrasonography and scintigraphy the effect of a somatostatin analog, octreotide, on gastric emptying and antral contraction. SUBJECTS: Twenty healthy men; mean age 23.9 years METHODS: Subjects were studied for 7 days, once after subcutaneous injection of octreotide, 50 microg, 5 min before the ingestion of a test meal and once after subcutaneous injection of placebo. Ten subjects received a liquid meal, 10 others received a solid meal. With the liquid meal, gastric emptying was measured 15 min after its ingestion and antral contraction was measured for 15 min by ultrasonography. With the solid meal, gastric emptying was measured both by ultrasonography (n = 10) and by simultaneous scintigraphy (n = 6), with antral contraction measured by ultrasonography for 5 h after ingestion of the meal. RESULTS AND CONCLUSIONS: Octreotide given with a liquid or solid meal inhibited gastric emptying in healthy subjects. A significant suppression of antral contraction occurred only with a solid meal.

Effect of prior O2 breathing on ventilatory response to sustained isocapnic hypoxia in adult humans.

Sixteen healthy volunteers breathed 100% O2 or room air for 10 min in random order, then their ventilatory response to sustained normocapnic hypoxia (80% arterial O2 saturation, as measured with a pulse oximeter) was studied for 20 min. In addition, to detect agents possibly responsible for the respiratory changes, blood plasma of 10 of the 16 subjects was chemically analyzed. 1) Preliminary O2 breathing uniformly and substantially augmented hypoxic ventilatory responses. 2) However, the profile of ventilatory response in terms of relative magnitude, i.e., biphasic hypoxic ventilatory depression, remained nearly unchanged. 3) Augmented ventilatory increment by prior O2 breathing was significantly correlated with increment in the plasma glutamine level. We conclude that preliminary O2 administration enhances hypoxic ventilatory response without affecting the biphasic response pattern and speculate that the excitatory amino acid neurotransmitter glutamate, possibly derived from augmented glutamine, may, at least in part, play a role in this ventilatory enhancement.

Incorporation of fatty acids by Streptococcus mutans.

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.

Immunotherapy with paternal lymphocytes preceding in vitro fertilization-embryo transfer for patients with repeated failure of embryo transfer.

Immunotherapy with paternal lymphocytes for unexplained recurrent aborters was applied, preceding IVF-ET, for infertile women with repeated failure of ET. In one patient showing close histocompatibility with the husband, the subsequent new IVF-ET was successful under positive MLR-blocking antibody induced by immunotherapy.