Faecal microbiota in dogs with multicentric lymphoma.

Malignant lymphoma B-cell type is the most common canine haematopoietic malignancy. Changes in intestinal microbiota have been implicated in few types of cancer in humans. The aim of this prospective and case-control study was to determine differences in faecal microbiota between healthy control dogs and dogs with multicentric lymphoma. Twelve dogs affected by multicentric, B-cell, stage III-IV lymphoma, and 21 healthy dogs were enrolled in the study. For each dog, faecal samples were analysed by Illumina sequencing of 16S rRNA genes and quantitative PCR (qPCR) for selected bacterial groups. Alpha diversity was significant lower in lymphoma dogs. Principal coordinate analysis plots showed different microbial clustering (P = .001) and linear discriminant analysis effect size revealed 28 differentially abundant bacterial groups in lymphoma and control dogs. The qPCR analysis showed significant lower abundance of Faecalibacterium spp. (q < .001), Fusobacterium spp. (q = .032), and Turicibacter spp. (q = .043) in dogs with lymphoma compared with control dogs. On the contrary, Streptococcus spp. was significantly higher in dogs with lymphoma (q = .041). The dysbiosis index was significantly higher (P < .0001) in dogs with lymphoma. In conclusion, both sequencing and qPCR analyses provided a global overview of faecal microbial communities and showed significant differences in the microbial communities of dogs presenting with multicentric lymphoma compared with healthy control dogs.

A dysbiosis index to assess microbial changes in fecal samples of dogs with chronic inflammatory enteropathy.

Recent studies have identified various bacterial groups that are altered in dogs with chronic inflammatory enteropathies (CE) compared to healthy dogs. The study aim was to use quantitative PCR (qPCR) assays to confirm these findings in a larger number of dogs, and to build a mathematical algorithm to report these microbiota changes as a dysbiosis index (DI). Fecal DNA from 95 healthy dogs and 106 dogs with histologically confirmed CE was analyzed. Samples were grouped into a training set and a validation set. Various mathematical models and combination of qPCR assays were evaluated to find a model with highest discriminatory power. The final qPCR panel consisted of eight bacterial groups: total bacteria, Faecalibacterium, Turicibacter, Escherichia coli, Streptococcus, Blautia, Fusobacterium and Clostridium hiranonis. The qPCR-based DI was built based on the nearest centroid classifier, and reports the degree of dysbiosis in a single numerical value that measures the closeness in the l2 - norm of the test sample to the mean prototype of each class. A negative DI indicates normobiosis, whereas a positive DI indicates dysbiosis. For a threshold of 0, the DI based on the combined dataset achieved 74% sensitivity and 95% specificity to separate healthy and CE dogs.

Clostridium perfringens enterotoxin and Clostridium difficile toxin A/B do not play a role in acute haemorrhagic diarrhoea syndrome in dogs.

Although an association between clostridial pathogens and canine idiopathic acute haemorrhagic diarrhoea syndrome (AHDS) has been described, the relevance of those bacteria and their toxins remains unclear. The aim of this study was to evaluate the association between severity of clinical signs and presence of Clostridium perfringens enterotoxin (CPE) and Clostridium difficile toxin A/B (CDT A/B) in faeces of dogs with AHDS. Faecal samples of 54 dogs with idiopathic AHDS were tested by qualitative CPE and CDT A/B ELISA, and PCR was performed to detect enterotoxin genes of C. perfringens (cpe) and toxin B genes of C. difficile (cdt b). Prevalence of cdt b and CDT A/B in dogs with AHDS was 10/54 and 2/54 versus 3/23 and 0/23 in control dogs. Prevalence of cpe was 35/54 in affected versus 9/23 in control dogs. Prevalence of CPE in dogs with AHDS (13/54) was higher compared with control dogs (0/23). No significant difference was detected between CPE-positive and -negative and between cpe-positive and -negative dogs in severity of clinical signs, duration of hospitalisation, mortality rate and selected laboratory parameters. This study suggests that CPE and CDT A/B do not play a role in idiopathic AHDS, are not associated with clinical parameters in affected dogs and cannot be used to predict disease outcome.

Differential inhibition of fibrinogen binding to agonist- and RGDS peptide-activated states of GPIIb-IIIa by an anti-GPIIIa monoclonal antibody, PMA5.

Platelet agonists and RGD-containing peptides can convert platelet membrane glycoprotein (GP) IIb-IIIa from its resting state to an activated state competent to bind soluble fibrinogen. We examined the effects of two anti-GPIIb-IIIa monoclonal antibodies, PMA1 and PMA5, on fibrinogen binding to agonist- and RGD-activated GPIIb-IIIa. PMA1 abolished aggregation of both agonist- and RGDS peptide-activated fixed platelets, and inhibited the binding of 125I-fibrinogen to these platelets almost completely. PMA5 had the same effects on agonist-activated platelets, but had little effect on the aggregation of RGDS-activated fixed platelets, and inhibited fibrinogen binding to RGDS-activated fixed platelets by only 44%. PMA5 bound to agonist- and RGDS-activated platelets equally. Immunoblot analysis showed that PMA5 bound to intact GPIIIa, but not to a 66 kDa fragment of GPIIIa digested by chymotrypsin. Although PMA5 inhibited platelet adhesion to immobilized fibrinogen by 94%, 44% of the remaining adherent platelets were spread. In contrast, no platelet spreading was observed in the presence of PMA1. These findings indicate that PMA5 is a novel anti-GPIIIa monoclonal antibody with the ability to inhibit fibrinogen binding to agonist- and RGD-activated states of GPIIb-IIIa differentially, and suggest that binding of immobilized fibrinogen to RGD-activated GPIIb-IIIa is necessary for platelet spreading.

Detection of platelet adhesion/aggregation to immobilized ligands on microbeads by an aggregometer.

Platelet aggregation is believed to follow platelet adhesion to vascular injury sites. We have developed a turbidimetric assay for platelet aggregation following platelet adhesion to immobilized ligands using an aggregometer. The addition of polystyrene beads coated with von Willebrand factor (vWF) or fibrinogen (Fg) to platelet suspensions caused prompt aggregation of beads and platelets, which was detected as an increase in light transmission. Electron microscopic analysis revealed that platelets adhered to the bead surfaces and that additional platelets adhered to already adhering platelets, leading to the formation of platelet aggregates. vWF-coated beads induced larger aggregates than Fg-coated beads. The interaction of vWF-coated beads with platelets was abolished by both GPIb and GPIIb-IIIa blockers, while that of Fg-coated beads was abolished by GPIIb-IIIa blockers. vWF-coated beads induced modest secretion of granules from platelets but no thromboxane B2 synthesis. Fg-coated beads induced neither reaction. However, pleckstrin phosphorylation and protein tyrosine phosphorylation was induced by both types of bead. Platelet aggregation following platelet adhesion to both types of bead was inhibited by ADP scavengers, a protein kinase C inhibitor and a tyrosine kinase inhibitor, but not by aspirin. These findings suggest that vWF- and Fg-coated beads can induce platelet aggregation following platelet adhesion through specific ligand-receptor interactions and intracellular signaling. Our simple assay using these beads may represent a useful test for immobilized ligand-induced platelet adhesion and aggregation.

A novel mechanism for exposure of fibrinogen binding sites on GPIIb-IIIa by a monoclonal antibody.

Conformational changes in platelet membrane glycoprotein (GP) IIb-IIIa, whose nature is not defined, lead to exposure of fibrinogen binding sites. We have reported previously that F(ab')2 fragments of a monoclonal antibody, PMA4, directed against the GPIIb-IIIa complex-specific domain, induced binding of fibrinogen to platelets without causing intracellular activation, whereas Fab did not. In this study, we examined the mechanism responsible for the difference in the ability of PMA4 F(ab')2 and Fab to expose fibrinogen binding sites. PMA4 Fab had affinity for GPIIb-IIIa similar to that of PMA4 F(ab')2. Addition of F(ab')2 goat anti-mouse Fab antibody to cross-link PMA4 Fab-bound GPIIb-IIIa molecules induced fibrinogen binding. There was a direct correlation between the number of molecules of PMA4 F(ab')2 and the amount of fibrinogen bound. PMA4 did not recognize ligand-induced binding sites (LIBS). These results suggest that the cross-linking of special sites on the GPIIb-IIIa complex-specific domain by bivalent antibody alters the conformation of GPIIb-IIIa to a state competent to bind soluble fibrinogen and that conformational changes in non-LIBS are involved in the mechanism for exposing fibrinogen binding sites on GPIIb-IIIa.

An alloantibody recognizing the FVIII A1 domain in a patient with CRM reduced haemophilia A due to deletion of a large portion of the A1 domain DNA sequence.

We report the development of a FVIII inhibitor in a patient with severe, cross reacting material reduced (CRM(R)) haemophilia A. The level of Factor VIII antigen (FVIII:Ag) measured by ELISA using anti-C2 monoclonal and alloantibodies was 1.9 U/dl. This baseline FVIII:Ag level was increased to 8.3 U/dl after administration of DDAVP. The anti-FVIII inhibitor titer was 2.9 Bethesda U/ml. DNA analysis showed a large deletion of the FVIII gene from exon 4 to 7, corresponding to amino acid residues 111-317 included within the A1 domain. The size of the gene deletion was approximately 28 kb. 5' and 3' breakpoints were identified by sequencing in intron 3 and intron 7, respectively. FVIII mRNA was detected in the patient's peripheral lymphocytes and the deletion spanning exon 4 to 7 was confirmed at the RNA level. Immunoprecipitation experiments using 125I labeled A1, A2 and light chain demonstrated that the inhibitor reacted only with the 54 kDa A1 domain. The inhibitor activity was more than 95% neutralized by A1 domain polypeptide. Our findings suggest a close relationship between the inhibitor epitope and the specific gene deletion with regard to the pathogenesis of the inhibitor in this patient.

Polymorphisms of HPA-1 through 6 on platelet membrane glycoprotein receptors are not a genetic risk factor for myocardial infarction in the Japanese population.

We examined HPA genotypes derived from polymorphisms of platelet membrane receptors in 88 Japanese patients with early-onset myocardial infarction and in 100 control subjects. The results indicated that HPA genotypes 1 through 6 are not associated with early-onset myocardial infarction.

Outside-in signaling from integrin alpha IIb beta 3 into platelets in the absence of agonist-induced signaling.

Platelet agonists generate intracellular signals which lead to activation of the platelet membrane glycoprotein IIb-IIIa (integrin alpha IIb beta 3). The resulting occupancy of alpha IIb beta 3 by ligands also generates signals into the cell (outside-in signaling). We reported previously that unlike platelet agonists, the F(ab')2 fragments of an anti-alpha IIb beta 3 monoclonal antibody, PMA4, induced fibrinogen binding to alpha IIb beta 3 without causing intracellular activation. In this study, in order to determine whether outside-in signaling occurs in the absence of agonist-induced intracellular signals, we used PMA4 F(ab')2 as an inducer of fibrinogen binding to alpha IIb beta 3. PMA4 F(ab')2-induced fibrinogen binding and subsequent platelet aggregation triggered tyrosine phosphorylation of several proteins including pp72syk but not pp125FAK. No Ca2+ influx or mobilization, thromboxane B2 synthesis, phosphorylation of pleckstrin or the myosin light chain, cytoplasmic alkalinization, or platelet shape changes, were detected. These findings suggest that, in the absence of agonist-induced signaling, alpha IIb beta 3 occupied by soluble fibrinogen generates only a limited outside-in signal.

Clinical trial to investigate the pharmacokinetics, pharmacodynamics, safety, and efficacy of recombinant factor VIIa in Japanese patients with hemophilia with inhibitors.

A multicenter and open-labeled clinical trial of human recombinant factor VIIa (rFVIIa) was conducted in Japanese patients with severe hemophilia A or B with inhibitors. The trial consisted of 2 parts. In study 1, the pharmacokinetics, pharmacodynamics, and safety of a single dose of 120 microg/kg of rFVIIa were investigated in 8 patients. In the subsequent study 2, the hemostatic effect and safety of rFVIIa were evaluated during a 24-week period in 10 patients. In study 1, the mean maximum FVII-coagulant activity (FVII:C) was found to occur after 10 minutes; activity then decreased rapidly and returned to the baseline within 24 hours after a single intravenous infusion of rFVIIa. The mean half-life of FVII:C was 3.5 hours. The activated partial thromboplastin time and prothrombin time in the patients were immediately shortened but returned to the baseline within 24 hours after dosing. In study 2, 86 microg/kg to 120 microg/kg of rFVIIa (mean, 97 microg/kg) was administered 1 to 85 times to 10 patients. A total of 58.0% (91/157) of bleeding episodes were treated excellently or effectively, with 5 (3.2%) ineffective episodes. There was no apparent trend in the relationship of the hemostatic effect with bleeding sites, mean dose, or number of injections. The efficacy rate, however, was significantly higher (90.0%) in bleeding episodes treated within 3 hours than in those treated at longer intervals (31.0%). No treatment-related adverse events were observed, and there was no evidence of antibody formation to rFVIIa. In conclusion. rFVIIa is an effective and well-tolerated option for treatment of bleeding episodes in hemophilia patients with inhibitors.

In vivo microdialysis of amino acid neurotransmitters in the hippocampus in amygdaloid kindled rat.

Extracellular concentrations of gamma-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) were determined by microdialysis in rat hippocampus during various amygdaloid kindled stages. The values of GABA and Glu were increased 3-4 times in C2-C3 stages in comparison with the values in control animals. After reaching the C5 stages, these values were increased 3-7 times. However, the concentration of Asp decreased depending on the kindling stage, reaching the lowest value of 33% in comparison with the normal value. The observed changes may be related to kindling induced seizures.

Neuroprotective effect of cholecystokininB receptor antagonist on ischemia-induced decrease in CA1 presynaptic fiber spikes in rat hippocampal slices.

The effects of cholecystokinin (CCK) receptor agonists and antagonists on hypoxia/hypoglycemia (ischemia)-induced decrease in CA1 presynaptic fiber spikes elicited by the stimulation of Schaffer collaterals were investigated using rat hippocampal slices. Treatment with the CCKB receptor agonist CCK tetrapeptide (CCK4, 0.01-10 microM) exacerbated the ischemia-induced decrease in the CA1 presynaptic potential in a concentration-dependent manner. Whereas, treatment with the CCKB receptor antagonist [(3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepin-3-yl)-N1-(3-methylphenyl)-urea] (L365260), and not with CCKA receptor antagonist [(3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepin-3-yl)-1H-indole-2-carboxamide] (L364718), produced a concentration-dependent attenuation of the ischemia-induced decrease. The magnitude of recovery of the CA1 field potentials in L365260-treated groups at 10 and 100 nM was 34% and 45%, respectively. The neuroprotective effect of L365260 (0.01 and 0.1 microM) was completely blocked by co-treatment with CCK4 (0.1 microM), a concentration that did not affect the decreased presynaptic potential induced by ischemia. These results demonstrated that the stimulation of the CCKB receptor played a detrimental role in the development of ischemic damage, whereas the blockade of CCKB receptors played a neuroprotective role in ischemic damage, suggesting a facilitatory role of CCK receptor-operated function in ischemia-induced neuronal deficits.

Aging impairs methamphetamine-induced free-running and anticipatory locomotor activity rhythms in rats.

In the present experiment, under drinking of 0.01% methamphetamine (MA), young rats exhibited free-running locomotor rhythm ranging from 24.6 to 30.9 h period under light-dark cycle. In contrast, 24-month-old rats showed a low activity and did not exhibit MA-induced free-running rhythm. When MA was injected at a fixed time of day (14:00), young rats exhibited intense locomotor activity from 1-2 h before drug-injection time. This preinjection activity was still detected on the withdrawal day. In addition, intense locomotor activity was observed for 12:00-17:00, even on the withdrawal day (MA-associated anticipatory activity). Thus, MA-associated activity of the withdrawal day became an another good index for MA anticipatory activity rhythms whereas old rats did not exhibit this anticipatory activity. The present study suggests that aging strongly impairs the manifestation of MA-induced free-running and anticipatory activity rhythms in rats.

Age-related impairment of food anticipatory locomotor activity in rats.

In rats of various ages (11 weeks to 24 months), restricted feeding was carried for 6 successive days and food was withheld completely on the 7th day to assess the effect of aging on food anticipatory activity. When feeding was restricted to a single meal at a fixed time of day (1330-1730), rats exhibited intense locomotor activity from 1-3 h before feeding time (prefeeding activity). This prefeeding activity was still detected on the fasting day. In addition, intense locomotor activity was observed for 1330-1730 even on the fasting day (mealtime-associated activity). Thus, mealtime-associated activity of the fasting day became an another good index for food anticipatory activity. These results indicate that the rats had come to anticipate the mealtime. Both prefeeding and mealtime-associated activities were similar at 11 weeks and 9 months of age, but they were impaired at 12 and 24 months of age. In 11-week-old rats, there was a negative correlation between the level of mealtime-associated activity and the daily duration of access to food, but this relationship was absent in 12-month-old rats. The present study suggests that aging strongly impairs the manifestation of food anticipatory activity in rats.

Effect of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 on food-anticipatory activity rhythm in the rat.

When feeding was restricted to a single meal scheduled at a fixed time (1300-1700 h) in the day, rats exhibited increased prefeeding locomotor activity 1-3 h before the feeding time, which increased over 6 days. The activity increase was seen for about 5 h (1100-1600 h) on the seventh day when no food was provided (fasting day), as the rat anticipated mealtime. Thus, mealtime-associated activity rhythm on the fasting day was considered to be another good index of food-anticipatory activity. In this study, we investigated the effects of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, which has been reported to cause impairment of spatial learning, on food-anticipatory activity in rats. Daily injection of MK-801 (0.2 mg/kg) at 1700 h for 6 successive days impaired the development of increased prefeeding activity. The appearance of mealtime-associated activity was impaired by daily injection of MK-801 (0.2 mg/kg) at 1700 h (immediately after food restriction, FR) or at 1900 h (2 h after FR) but not at 0900 (16 h after FR). In addition, mealtime-associated activity was impaired by MK-801 injected at 1700 h in a dose-dependent manner (0.1-1 mg/kg). The present results demonstrate that NMDA receptor mechanisms were involved in learning processes of food anticipation and suggest that an impairment of food-anticipatory activity may be considered an animal model of a deficit of "temporal learning".

Attenuating effect of arecoline and physostigmine on an impairment of mealtime-associated activity rhythm in old rats.

In the present study, we examined whether cholinergic drugs such as arecoline and physostigmine attenuated an impairment of time perception presented by daily scheduled feeding in aged rats. When feeding was restricted to a single meal at a fixed time of day (13:00-17:00) for 6 successive days, young rats exhibited intense locomotor activity from 1-3 h before feeding time. Intense locomotor activity was observed between 12:00-17:00 in young animals even on the fasting day (on day 7) (mealtime-associated activity). However, this mealtime-associated activity was impaired in old rats. Daily injection of arecoline (10 mg/kg) or physostigmine (0.1 and 0.2 mg/kg) at 17:00 for 6 successive days attenuated the impairment of mealtime-associated activity on the fasting day in a dose-dependent manner in old rats, whereas daily treatment with D-glucose (100 or 2000 mg/kg) did not. The results of the present study suggest that cholinergic drugs attenuate the impairment of the manifestation of mealtime-associated anticipatory activity related to 'temporal learning' in old rats.

Attenuating effect of bifemelane on an impairment of mealtime-associated activity rhythm in aged and MK-801-treated rats.

In the present experiment, we examined the attenuating effect of bifemelane hydrochloride (BF), 4-(o-benzyl phenoxy)-N-methylbutylamine hydrochloride, on the impairment of time perception caused by daily scheduled feeding using aged and MK-801-treated rats. When feeding was restricted to a single meal at a fixed time of day (1300-1700 h) for six successive days, young rats exhibited intense locomotor activity 1-3 h before feeding time. Intense locomotor activity was observed for 1200-1700 h even on the fasting day (day 7; mealtime-associated activity). Mealtime-associated activity was impaired in 24-mo-old rats and also in N-methyl-D-aspartate receptor antagonist, MK-801-treated rats. Daily injections of bifemelane at 1700 h for six successive days significantly attenuated the impairment of mealtime-associated activity on the seventh day in a dose-dependent manner in aged rats. In addition, cotreatment of MK-801 with bifemelane blocked the MK-801-induced impairment of mealtime-associated activity. The present study suggests that bifemelane has an enhancing effect on learning and memory performance, such as spatial and temporal perception.

Attenuating effect of serotonin receptor antagonists on impairment of mealtime-associated activity rhythm in old rats.

In the present study, we examined attenuating effect of serotonin (5-HT) receptor antagonists on the impairment of the time perception presented by daily scheduled feeding in old rats. When feeding was restricted to a single meal at a fixed time of day (1300-1700 h) for six consecutive days, young rats exhibited intense locomotor activity from 1-3 h before feeding time. Intense locomotor activity was observed between 1200 adn 1700 h in young animals even on the fasting day (day 7) (mealtime-associated activity). However, this mealtime-associated activity was impaired in 24-mo-old rats. Daily injections of 5-HT2 receptor antagonists, mianserin or ritanserin, or a 5-HT3 receptor antagonist, Y25130, at 1700 h for 6 consecutive days significantly and dose-dependently attenuated the impairment of mealtime-associated activity on the fasting day in old rats without affecting the food intake. Our results suggest that the blockade of 5-HT2 and/or 5-HT3 receptors attenuates impairment of the manifestation of mealtime-associated anticipatory activity related to temporal learning in old rats.

[Reducing cross-talk in electrooculograms made with several electrodes].

In order to discuss how to handle the contralateral effect (cross-talk) when taking an electrooculogram (EOG) with two or more electrodes, a forced duction test was used to detect cross-talk potentials. The cross-talk potentials originating from the left eye were measured at the right eye for 5 subjects when the left eye was adducted and abducted alternately with a suction contact lens while the right eye was kept fixed. The cross-talk potentials detected at the right eye did not depend on the electrode position, but the contralateral EOG potentials depended on the electrode position around the left eye. The cross-talk value is calculated as the gain, 20.log [(cross-talk potential)/(contralateral EOG potential)]. The denominator of this expression considerably affects the gain since the contralateral potential depends on the position of the electrode. We propose that the effect of the cross-talk on the potential between a pair of electrodes can be cancelled with the gain value (-15.0dB: the mean value for the 5 subjects) when the contralateral electrode is set 20 mm from the outer canthus.

[Inversion of chromosome 16 observed in acute myeloblastic leukemia (M2) with biphenotypic surface markers lacking monocytosis and eosinophilia].

Inversion of chromosome 16 was found in a 73-year-old female with acute myeloblastic leukemia (FAB:M2). Complete remission was achieved by combined chemotherapy (DNR, Ara-C, 6-MP, Prednisolone), but she relapsed 6 months later without CNS involvement and died of respiratory failure presumably due to cerebrovascular accident during remission reinduction chemotherapy. Biphenotypic surface markers (CD2+ and CD13+) were observed on relapse. Eosinophilia was not observed throughout. Our patient and the other reported case suggest that biphenotypism and the lack of eosinophilia and monocytosis in inv (16) leukemia may be correlated with a poor prognosis.