Differential production of MCP-1 and cytokine-induced neutrophil chemoattractant in the ischemic brain after transient focal ischemia in rats.

Chemokines have been shown to play an important role in leukocyte infiltration into ischemic lesions. Recently, the increased expression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC) was observed in experimental stroke models where infiltrated leukocytes were supposed to induce tissue injury, however, the protein level and time course of these chemokines have not been fully elucidated. Therefore, we analyzed the time-dependent production of MCP-1 and CINC in the rat brain after transient middle cerebral artery occlusion (MCAO) by means of specific enzyme-linked immunosorbent assay systems. The MCP-1 levels in the ipsilateral hemispheres increased from 6 h, peaked at 2 days, and thereafter gradually decreased. The peak MCP-1 concentration was 89.2+/-28.2 ng/g tissue wet weight (mean +/- SEM, n = 5, 49.3-fold greater than the contralateral value at the same time, P < 0.05), which is supposed to be high enough to exert its biological effects. In contrast, the maximum CINC concentration that corresponded to 2.9+/-0.7 ng/g tissue wet weight (mean +/- SEM, n = 5, 55.0-fold greater than the contralateral value at the same time, P < 0.05), was observed at 6 h. In addition, we confirmed the temporal profile of leukocyte subtypes that infiltrated into the ischemic brain, thus, neutrophil infiltration occurred at early stages (1-3 days), followed by massive infiltration of macrophages at later stages (2-7 days). These studies suggest that MCP-1 in cerebral ischemia actually plays a significant role in the migration of macrophages into the lesion and that the differential temporal production of these chemokines contributes to the regulation of infiltrated leukocyte subtypes.

Effects of a highly selective synthetic inhibitor of plasma kallikrein on disseminated intravascular coagulation in rats.

We found a new, highly selective plasma kallikrein inhibitor, trans-4-aminomethyl-cyclohexanecarbonylphenylalanine 4-carboxymethylanilide hydrochloride, called PKSI-527 in our laboratories. This study was conducted to evaluate PKSI-527, on thromboplastin (TP)- and endotoxin (LPS)-induced disseminated intravascular coagulation (DIC) in rats. PKSI-527 was infused intravenously at 0.1 mg/kg/min for 250 min. Three of the parameters of the coagulation and fibrinolysis system, fibrinogen level, platelet counts and fibrin(ogen) degradation products (FDP) level were assayed. PKSI-527 prevented the change in the coagulation and fibrinolysis system in LPS-induced DIC, however it was not clearly effective in TP-induced DIC. The parameters of organ failure, such as serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatine phosphokinase (CPK), lactate, blood urea nitrogen and beta-glucuronidase, were assayed. Although the changes in the fibrinogen level, platelet counts and FDP level were almost the same in both models, the parameters of organ failure apparently increased in LPS-induced DIC more so than in TP-induced DIC. PKSI-527 significantly suppressed the increases in GOT and GPT in LPS-induced DIC. These results indicate that plasma kallikrein may play a significant role in LPS-induced DIC. Therefore, PKSI-527, as a synthetic plasma kallikrein inhibitor may be a valuable tool to explore the mechanism of DIC and the accompanying organ failure.

Characteristic effects of activated human protein C on tissue thromboplastin-induced disseminated intravascular coagulation in rabbits.

Protein C (PC) is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating thrombosis and fibrinolysis by inhibiting not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). In the present study we examined the effects of human APC on tissue thromboplastin-induced disseminated intravascular coagulation (DIC) in rabbits and compared them with those of heparin. Both APC (300-3000 U/kg) and heparin (100-300 IU/kg) inhibited the decreases in platelet count and fibrinogen level equally. APC improved the prolonged bleeding time, but heparin aggravated bleeding with potent prolongation of activated partial thromboplastin time (APTT). Furthermore, in APC-treated animals, fibrin deposition in glomeruli was less than in heparin-treated animals. This result that APC accelerated local fibrinolysis by neutralizing PAI-1. From our findings, we concluded that APC can improve both coagulation and fibrinolysis in a DIC model and should be useful for the clinical remedy of DIC without having an adverse side effect like a bleeding tendency.

Species specificity of anticoagulant activity of activated human protein C: involvement of factor V as well as protein S.

Activated protein C (APC) possesses species specificity in its anticoagulant activity. Human APC exerts only weak activity in rat plasma compared with that in human plasma. The present study was undertaken to estimate the difference in interaction of human and rat factors with human APC and to assess the cause of the species specificity. Human or rat protein S (PS), factor V, or factor VIII was used to supplement human plasma depleted of each respective factor, and the anticoagulant activity of human APC was measured in term of the elongation of activated partial thromboplastin time (APTT). The activity of human APC in rat PS- or factor V-supplemented plasma was weaker than that in the human PS- or factor V-supplemented plasma. Furthermore, using purified human and rat factor V, human APC showed weaker inactivation of rat factor V than human factor V. Equal anticoagulant activity was observed in human or rat factor VIII-supplemented plasma. And there was a little difference in the interaction of APC with its inhibitors in human or rat plasma during a few minutes of incubation as judged by measurement of residual activity by an enzyme capture assay. From these results factor V as well as PS seems to play a major role in the species specificity of APC.

[An experimental study of the diurnal changes in colonic motility centering on defecation].

The following findings have been obtained as a result of making an assessment regarding the diurnal changes in colonic motility by means of continuous measurement of contractile waves by using strain gauge force transducers and roentgenographic observation in conscious dogs. 1. Before defecation, the contractile force of the wave was weak, frequency of its emergence was also small, and transfer of intestinal content was slow, showing decrease of colonic motility. 2. After defecation, the gradually increasing and decreasing contractile wave groups became clear, and the contractile force was intensified concurrently with increase of its emerging frequency. Transfer of intestinal content to the anal side was rapid, and recovery of colonic motility was observed. 3. The recovery of the colonic motility after defecation was observed regardless of digestive or interdigestive state. 4. By intake of food, increase of the colonic motility corresponding to gastrocolic response was observed, but it was due to the increase of emerging frequency of contractile wave, for which no change was observed in contractile force or duration in each individual waves. 5. It was suggested that the contractile motion which undergoes gradual increase and decrease is the basic pattern in the colonic motility and that the colonic motility changes by the differences of amount, shape and hardness of intestinal content, and decreases gradually along with increase of intestinal content, but the basic pattern of contractile motion is restored by inflow of intestinal content into the colon which became empty after defecation. From the above it was considered to be inadequate to use the pattern classification of digestive and interdigestive state for the analysis of colonic motility and that assessments should be made centering on defecation.

Effects of progesterone on the elevation of tail skin temperature in ovariectomized rats.

We examined the effects of progesterone on the elevation of tail skin temperature (TST) in ovariectomized rats and compared them with those of estradiol. Progesterone showed only insignificant effects on the TST elevation, whereas estradiol showed complete inhibition. The TST elevation induced by ovariectomy is caused by estradiol deficiency, but progesterone plays little or no role.

Primary Sjögren's syndrome complicated by sarcoidosis.

Sjögren's syndrome and sarcoidosis share several common features, such as keratoconjunctivitis sicca, swelling of parotid glands, lung involvement, cutaneous anergy, T cell-mediated immunodeficiency, an increased CD4+/CD8+ lymphocyte ratio, and association with the human leucocyte antigen (HLA)-B 8 and DR 3 haplotypes. However, only five patients with primary Sjögren's syndrome and sarcoidosis have been previously reported in the English language literature. The rare case of a 49-year-old Japanese woman with primary Sjögren's syndrome complicated by sarcoidosis is described. The serum angiotensin-converting enzyme level was increased, and histological examination of lung and skin biopsies revealed noncaseating granulomas, indicating that her primary Sjögren's syndrome was complicated by sarcoidosis.

[A simple method of extracorporeal membrane oxygenation (ECMO)--2: The effects of ECMO (arterio-venous shunt) on the arterial blood gases measured for 24 hours].

In eight anesthetized mongrel dogs with thiopental, diazepam and pancuronium bromide, ECMO with arterio-venous shunt was performed under hypoventilation for 24 hours. The blood flow through the ECMO which was connected between the femoral artery and vein was approximately 40% of the cardiac output. Hypoventilation was induced to decrease PaO2 to around 40mmHg and increase PaCO2 to around 80 mmHg. The ECMO improved the abnormal parameters, which were induced by the hypoventilation, to almost normal range (PaO2 80mmHg, PaCO2 30mmHg) for 24 hours. Furthermore, there were no abnormalities in cardiovascular and other systems during ECMO.

VNTR polymorphism of the collagen type II, alpha 1 (COL2A1) gene detected by PCR.

The 3' side of the human type II collagen alpha 1 (COL2A1) gene contains a region consisting of a variable number of tandemly repeated short A + T-rich DNA sequences (VNTR). We amplified this region accurately by the polymerase chain reaction (PCR). Genomic DNA was purified from isolated buffy-coat cells, and thermostable Taq polymerase was used to amplify the target region. The amplification products were directly visualized after polyacrylamide gel electrophoresis. In this way, five alleles were distinguished in chromosomes from 33 unrelated Japanese, and named A, B, C, D, and E in decreasing order of length. The relative frequencies of the COL2A1 3' VNTR alleles A through E were 0.045, 0.075, 0.469, 0.015, and 0.393, respectively. Co-dominant segregation was observed in two informative families. The COLA2A1 3' VNTR locus was estimated to have a heterozygosity index of 62% and a polymorphic information content of 0.55.

Mu opioid receptor antagonism in the nucleus accumbens shell blocks consumption of a preferred sucrose solution in an anticipatory contrast paradigm.

Binge eating, a central feature of multiple eating disorders, is characterized by excessive consumption occurring during discrete, often brief, intervals. Highly palatable foods play an important role in these binge episodes - foods chosen during bingeing are typically higher in fat or sugar than those normally consumed. Multiple lines of evidence suggest a central role for signaling by endogenous opioids in promoting palatability-driven eating. This role extends to binge-like feeding studied in animal models, which is reduced by administration of opioid antagonists. However, the neural circuits and specific opioid receptors mediating these effects are not fully understood. In the present experiments, we tested the hypothesis that endogenous opioid signaling in the nucleus accumbens promotes consumption in a model of binge eating. We used an anticipatory contrast paradigm in which separate groups of rats were presented sequentially with 4% sucrose and then either 20% or 0% sucrose solutions. In rats presented with 4% and then 20% sucrose, daily training in this paradigm produced robust intake of 20% sucrose, preceded by learned hypophagia during access to 4% sucrose. We tested the effects of site-specific infusions of naltrexone (a nonspecific opioid receptor antagonist: 0, 1, 10, and 50µg/side in the nucleus accumbens core and shell), naltrindole (a delta opioid receptor antagonist: 0, 0.5, 5, and 10µg/side in the nucleus accumbens shell) and beta-funaltrexamine (a mu opioid receptor antagonist: 0 and 2.5µg/side in the nucleus accumbens shell) on consumption in this contrast paradigm. Our results show that signaling through the mu opioid receptor in the nucleus accumbens shell is dynamically modulated during formation of learned food preferences, and promotes binge-like consumption of palatable foods based on these learned preferences.

Convergent, not serial, striatal and pallidal circuits regulate opioid-induced food intake.

Mu opioid receptor (MOR) signaling in the nucleus accumbens (NAcc) elicits marked increases in the consumption of palatable tastants. However, the mechanism and circuitry underlying this effect are not fully understood. Multiple downstream target regions have been implicated in mediating this effect but the role of the ventral pallidum (VP), a primary target of NAcc efferents, has not been well defined. To probe the mechanisms underlying increased consumption, we identified behavioral changes in rats' licking patterns following NAcc MOR stimulation. Because the temporal structure of licking reflects the physiological substrates modulating consumption, these measures provide a useful tool in dissecting the cause of increased consumption following NAcc MOR stimulation. Next, we used a combination of pharmacological inactivation and lesions to define the role of the VP in hyperphagia following infusion of the MOR-specific agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) into the NAcc. In agreement with previous studies, results from lick microstructure analysis suggest that NAcc MOR stimulation augments intake through a palatability-driven mechanism. Our results also demonstrate an important role for the VP in normal feeding behavior: pharmacological inactivation of the VP suppresses baseline and NAcc DAMGO-induced consumption. However, this interaction does not occur through a serial circuit requiring direct projections from the NAcc to the VP. Rather, our results indicate that NAcc and VP circuits converge on a common downstream target that regulates food intake.

Portal systemic shunt through the renal vein.

BACKGROUND: Despite the semi-routine use of color Doppler sonography for evaluating portal circulation abnormalities, there is a relative paucity of detailed color Doppler findings of portal systemic (P-S) shunt through the renal vein (P-SR shunt). METHODS: We reviewed the color Doppler findings of 18 patients with P-SR shunt to determine its clinical significance and appropriate scanning techniques for diagnosing accurately P-SR shunt. RESULTS: The splenorenal shunt was imaged as a highly tortuous vessel at the splenic hilum, which then coursed backward behind the spleen. Splenic vein flow was reversed or very slow. The gastrorenal shunt originated from the splenic vein, coursed backward, and joined the left renal vein. Flow direction in the splenic vein was always hepatopetal. The P-S shunt through the right renal vein originated from duodenal or jejunal varices, coursed posterolaterally, and joined the right renal vein at the renal hilum. CONCLUSION: Familiarity with these color Doppler findings will help increase the diagnostic confidence of P-SR shunt by color Doppler sonography.

Malignant fibrous histiocytoma of the spleen: report of a case and literature review.

There is a marked paucity of reports on malignant fibrous histiocytoma (MFH) of the spleen in the literature, and there are no previous reports of its color Doppler sonographic (US) and contrast-enhanced US findings. We report on an 82-year-old male with splenic MFH (inflammatory subtype), with an emphasis on color Doppler and contrast-enhanced US findings.

[Anti-thrombotic effect of activated protein C].

Protein C (PC) is an important anticoagulant protein in blood and converted to its active form, activated protein C (APC), by thrombin bound with thrombomodulin. APC exhibits an anticoagulant effect by the inactivation of FV a and FVIII a. In addition, APC exerts a profibrinolytic effect by inactivation of PAI-1 and inhibition of TAFI activation. APC is strongly anti-thrombotic because of its anticoagulant and profibrinolytic effect. APC has gamma-carboxyglutamic acid residues that bind to acidic phospholipids expressed on activated platelet or injured endothelial cells. Thus APC works only at the site where clots are formed and has a weak effect in primary hemostasis; this means that the use of APC is expected not to have any hemorrhagic risk. In both DIC animal models and clinical studies, we confirmed safer amelioration by APC than heparin. Recently, a specific receptor for PC/APC was found on endothelial cell membrane and anti-inflammatory effects of APC were also reported. Thus APC is thought to play an important regulatory role in blood coagulation, fibrinolysis and inflammation, especially in thrombotic diseases.

Comparison of hemorrhagic effect of heparin and human activated protein C with use of Thrombostat 4000.

The importance of bleeding as a complication of anticoagulant therapy is clearly recognized. We previously reported that amelioration of hemorrhage associated with disseminated intravascular coagulation by the human activated protein C (APC) was greater than that by heparin. In this study, we compared the bleeding complication of intravenously administered APC and heparin in rabbits, and also estimated primary hemostasis. When both anticoagulants were intravenously infused, the bleeding time from a punctured ear vein was prolonged dose-dependently. However, at doses which prolonged the activated partial thromboplastin time nearly equally, the prolongation of bleeding was greater in heparin-administered rabbits. Blood withdrawn from heparin-administered animals showed increases in in vitro bleeding parameters which correlated with the in vivo bleeding time. However, only small changes were observed in the blood withdrawn from APC-administered animals. Both drugs induced either no change or only a slight decrease in the platelet count, hematocrit and fibrinogen content. These observations suggest that APC may be a more useful anticoagulant than heparin since it causes less bleeding tendency.

Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo.

The endothelial cell protein C receptor (EPCR) facilitates protein C activation by the thrombin-thrombomodulin complex. Protein C activation has been shown to be critical to the host defense against septic shock. In cell culture, tumor necrosis factor-alpha (TNF-alpha) down-regulates EPCR expression, raising the possibility that EPCR might be down-regulated in septic shock. We examined EPCR mRNA and soluble EPCR levels in mice and rats challenged with lethal dose 95 levels of endotoxin. Toxic doses of TNF-alpha failed to alter EPCR mRNA levels in mice. Rather than EPCR mRNA levels falling in response to endotoxin, as predicted from cell-culture experiments, they rose approximately 3-fold 6 hours after exposure to endotoxin before returning toward baseline levels at 24 hours after exposure. Soluble EPCR levels rose approximately 4-fold. Infusion of hirudin, a specific thrombin inhibitor, before endotoxin exposure almost completely blocked the increase in EPCR mRNA and soluble EPCR. Consistent with the idea that the responses were mediated by thrombin, thrombin infusion (5 U/kg of body weight for 3 hours) resulted in an approximately 2-fold increase in EPCR mRNA and soluble EPCR. Incubation of rat endothelial cells with thrombin or murine protease-activated receptor 1 agonist peptide resulted in a 2-fold increase in EPCR mRNA. These results indicate that thrombin plays a major role in up-regulating EPCR mRNA and shedding in vivo. (Blood. 2000;95:1687-1693)

A finding of highly selective synthetic inhibitor of plasma kallikrein; its action to bradykinin generation, intrinsic coagulation and experimental DIC.

We found a novel and highly selective synthetic inhibitor of plasma kallikrein (PK), called PKSI-527; the Ki value was 0.81 microM. PKSI-527 inhibited the bradykinin (BK) generation induced by kaolin and prolonged partial thromboplastin time (PTT). PKSI-527 prevented the decrease of fibrinogen (Fg) levels due to i.v. injection of ellagic acid in mice and ameliorated the endotoxin (ET)-induced DIC in rats.

Effect of activated human protein C on disseminated intravascular coagulation induced by lipopolysaccharide in rats.

Protein C is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating coagulation and fibrinolysis by inactivating not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). The aim of the present study was to examine the effect of a human APC product (designated as CTC-111), compared with that of heparin, on the disseminated intravascular coagulation (DIC) induced by lipopolysaccharide (LPS) in rats. LPS (1 mg/kg/h) infusion was performed through a femoral vein for 4 h. One-fifth amount of the total dosage of CTC-111 or heparin was injected into the other femoral vein, followed by a 4-h infusion of the remainder. Both CTC-111 (10,000-100,000 U/kg) and heparin (400-800 IU/kg) inhibited the decrease in platelet count and fibrinogen level equally. The prolonged activated partial thromboplastin time and prothrombin time observed in DIC rats were further elongated in both CTC-111- and heparin-treated rats. But, this prolongation was less in CTC-111-treated rats than in the heparin-treated ones. Heparin inhibited the increase in fibrin and fibrinogen degradation products more prominently than CTC-111. On the other hand, CTC-111 strongly inhibited the increase in PAI-1 activity but heparin did not. These results suggest that CTC-111 may enhance fibrinolysis through its direct inhibitory effect on PAI-1. The parameters for liver or renal damage, i.e., plasma glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), creatinine (Cre) and blood urea nitrogen (BUN), were significantly increased by LPS infusion. Both CTC-111 (100,000 U/kg) and heparin (800 IU/kg) decreased the increase in GOT and GPT levels significantly, whereas neither affected the increase in Cre or BUN. From these results, the activation of the blood coagulation system might partially contribute to the progression of liver damage caused by LPS, and might be less involved in the progression of renal damage in this model. In conclusion, CTC-111 showed both anticoagulant and profibrinolytic activity in the LPS-induced DIC model without excessive prolongation of coagulation time. From these results, CTC-111 is expected to be a useful remedy for DIC without the risk of bleeding.