Integrin alphavbeta3 regulates microfibril assembly in human periodontal ligament cells.

Fibrillin-1 is the major structural component of extracellular microfibrils. However, the mechanism by which extracellular fibrillin-1 assembles into microfibrils is not fully understood. Fibrillin-1 contains the Arg-Gly-Asp (RGD) motif, which may allow binding to RGD-recognizing integrins. We hypothesized that integrin alphavbeta3 on the cell surface of human periodontal ligament (PDL) fibroblasts may influence fibrillin-1 assembly into cell/matrix layers. We treated PDL fibroblasts with an integrin alphavbeta3-specific antagonist to examine fibrillin-1 assembly. Western blotting and immunofluorescence analysis showed that treatment with the integrin alphavbeta3 antagonist at 5 muM clearly abolished fibrillin-1 deposition. These results provide for the first time evidence that integrin alphavbeta3 regulates extracellular assembly of fibrillin-1, thereby modulating cell-mediated homeostasis of microfibrils.

A novel therapeutic approach combining human plasma-derived Factors VIIa and X for haemophiliacs with inhibitors: evidence of a higher thrombin generation rate in vitro and more sustained haemostatic activity in vivo than obtained with Factor VIIa alone.

BACKGROUND AND OBJECTIVES: Therapy with recombinant Factor VIIa (rFVIIa) for haemophiliacs with inhibitors still has some unresolved problems, such as the requirement for frequent infusions of rFVIIa every 2-3 h to sustain haemostatic activity for an extended time-period and that the therapeutic dose of rFVIIa is not always predictable. In the present study, we searched for an effective combination of plasma-derived FVIIa with other blood coagulation factors, and demonstrated that a therapeutic approach combining plasma-derived FVIIa and Factor X (FX) was more useful for treating haemophiliacs with inhibitors than FVIIa alone. MATERIALS AND METHODS: The haemostatic effects of FVIIa and FX were evaluated in vitro and in vivo. In in vitro experiments we assessed the following: the ability to enhance the thrombin generation rate in a reconstituted blood coagulation model without Factor VIII (FVIII) or Factor IX (FIX); the ability to correct the activated partial prothrombin time (APTT) of FVIII-depleted plasma or FIX-depleted plasma; and the ability to correct the clotting time of haemophilia-like whole blood using thromboelastography (TEG). In in vivo experiments, the haemostatic activity of the combination treatment of FVIIa and FX was determined by measuring the bleeding time and TEG using a monkey haemophilia B model produced by the injection of anti-human FIX polyclonal antibodies. The degree of thrombogenicity of the combination was evaluated using the rabbit stasis model. RESULTS: The addition of FX to FVIIa dramatically enhanced the thrombin generation rate in the reconstituted blood coagulation model and corrected the prolonged APTTs of FVIII- and FIX-depleted plasmas to levels achieved by the replacement therapies. In contrast, the addition of prothrombin to FVIIa did not show such enhancing activity. Furthermore, FVIIa-induced whole blood clotting times in the FVIII- and FIX-inhibited states were also shortened by the addition of FX in a concentration-dependent manner. Finally, the co-administration of FVIIa (80 microg/kg) and FX (800 microg/kg) in a monkey haemophilia B model resulted in a more robust and persistent haemostatic effect on the secondary bleeding time and whole-blood clotting time of TEG than that of FVIIa alone. The results of rabbit stasis tests for evaluating the risk of thrombogenicity showed that the combination of FVIIa and FX was less thrombogenic than FEIBA. CONCLUSIONS: The present study demonstrated that the combination of FVIIa and FX appeared to have a higher and more sustainable haemostatic potential than FVIIa alone, and less thrombogenicity than FEIBA. A therapeutic approach combining FVIIa and FX could be a promising and novel approach to compensate for the disadvantages of rFVIIa and FEIBA for haemophiliacs with inhibitors.

Induction of acquired factor IX inhibitors in cynomolgus monkey (Macaca fascicularis): a new primate model of hemophilia B.

Inherited hemophilia dog and other transient hemophilic animal models have been used for evaluation of hemostatic agents for use in treatment of hemophilia. We established the first nonhuman primate hemophilic model by immunizing cynomolgus monkeys with human FIX (hFIX) in adjuvants. FIX activities of all three hFIX-immunized monkeys decreased transiently to less than 10% in accordance with prolongation of activated partial thromboplastin time (APTT). Forty micrograms of human factor VIIa (hFVIIa) per kilogram body weight (that was reported to be clinically effective) was administered to the monkey with the highest inhibitor titer to evaluate its usefulness as a hemophilia inhibitor model. Results of thromboelastography (TEG) after the injection demonstrated that the hemostatic effect of FVIIa in this model would be similar to that in hemophiliacs with inhibitors. The antibodies purified from the monkey's plasma by hFIX-immobilized gel were composed of two types: Ca(2+)-dependent and -independent antibodies, with features of IgG(1) and IgG(4). Both types of antibodies reacted to cynomolgus FIX, and only Ca(2+)-dependent antibodies also expressed inhibitory activity against cynomolgus FIX. Immunoblotting analyses of Ca(2+)-dependent antibodies using hFIX and its derivatives suggested that they recognized the Ca(2+)-dependent conformation related to the gamma-carboxyglutamic acid (Gla) domain. Comparison of FIX cDNA from human, cynomolgus monkey, and other species, and the results of immunization of various animals (goats, beagle dogs, rabbits, and rats) with hFIX in adjuvants strongly suggested that the development of acquired FIX inhibitors in the monkeys might be due to high cross-reactivity of the antibodies to molecular mimic antigens, hFIX, and cynomolgus FIX.

A rapid latex agglutination assay for the detection of penicillin-binding protein 2'.

A simple and rapid slide latex agglutination assay was developed to detect penicillin-binding protein 2' (PBP2') from isolates of staphylococi. PBP2' present in the membranes of methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant coagulase negative staphylococci (MRCNS) was rapidly extracted by alkaline treatment and, by combining with a slide agglutination reaction using latex particles sensitized with monoclonal antibodies raised against it, PBP2' could be detected from a single loopful of cells taken from agar plates not containing beta-lactum antibiotics within 15 min. In a study of clinical isolates previously characterized as either MRSA or methicillin-susceptible Staphylococcus aureus (MSSA) by antibiotic susceptibility testing, 231 specimens of 232 MRSA were PBP2' positive by latex agglutination, and the 87 specimens of MSSA were all negative. One specimen identified as MRSA by susceptibility testing but PBP2' negative by latex agglutination was confirmed as mecA gene negative by PCR. This simple and rapid slide latex reagent should be useful in clinical diagnostics.

Role of IgA, IgG, and IgM antibodies against Haemophilus parainfluenzae antigens in IgA nephropathy.

We have recently demonstrated glomerular deposition of outer membranes of Haemophilus parainfluenzae (HP) antigens (OMHP) and the presence of IgA antibody against OMHP in patients with IgA nephropathy (IgA-N). In this study, we analyzed IgA-, IgG-, and IgM-classes of antibodies against OMHP, and the relationship between these antibodies and renal lesions in IgA-N. The subjects included 44 patients with IgA-N and 62 patients with outer glomerular diseases (OGD); the latter group consisted of 23 patients with predominantly IgG or IgM deposits and small amounts of IgA in the mesangium (group A), and 39 with IgG or IgM deposits without IgA (group B). IgA, IgG, and IgM antibodies against OMHP in patients sera were detected by enzyme-linked immunosorbent assay (ELISA). Immunoblotting demonstrated that the IgA, IgG, and IgM antibodies against OMHP in the sera of IgA-N patients bound to components of OMHP with molecular weights of 19.5, 30, and 40.5 kD. The amino acid compositions of these three OMHP components were similar to those reported for the outer membrane protein (OMP) P6 precursor, OMP P5, and OMP P2 (porin) of Haemophilus influenzae. Both IgA-N and group A patients, (i.e. those with IgA-related renal disease), demonstrated a significantly higher level of IgA antibodies against OMHP than did group B patients. However, only IgA-N patients revealed a significant correlation between the IgA-antibody titer and degree of glomerular changes. IgA-N patients with macroscopic hematuria or arterio(lo)sclerosis had a significantly higher IgA antibody titer than other IgA-N patients. There was no relationship between renal lesions and IgG or IgM antibody titers in any group. These findings suggest that IgA antibodies against OMHP are significantly increased in patients with IgA-related renal disease compared to those without mesangial IgA deposits and that a significant relationship between these antibodies and renal lesions exists only in patients with IgA-N.

Circulating IgA, IgG, and IgM class antibody against Haemophilus parainfluenzae antigens in patients with IgA nephropathy.

We previously demonstrated a close relationship between the outer membranes of Haemophilus parainfluenzae (HP) antigens (OMHP) and IgA nephropathy (IgAN). Our objective was to clarify the relationship among IgA, IgG, and IgM class antibody against OMHP in the sera of 44 patients with IgAN and 62 patients with other glomerular diseases (OGD) by ELISA. Patients with IgAN showed a significantly higher level of IgA antibodies (P less than 0.0005) and IgG antibodies (P less than 0.001) against OHMP, than did patients with OGD. Positive correlations were observed between IgA and IgG antibodies, between IgA and IgM antibodies, and between IgG and IgM antibodies against OMHP in the sera of patients with IgAN. Immunoblotting showed that IgA, IgG, or IgM antibodies against OMHP in the sera of all patients with IgAN bound to components of OMHP. Amino acid sequences of three components of OMHP recognized by the sera from patients with IgAN revealed homology with those reported for outer membrane protein (OMP) P6 precursor, OMP P5, and P2 porin protein of H. influenzae. Results suggest that patients with IgAN have glomerular deposits of OMP P6 precursor, OMP P5, or P2 porin protein of HP, and a specific increase in the production of IgA antibodies against OMHP via polyclonal activation against these, with switching of production from one isotype to another, e.g. from IgM to IgA.

Haemophilus parainfluenzae antigen and antibody in renal biopsy samples and serum of patients with IgA nephropathy.

IgA nephropathy may be associated with colonisation with Haemophilus parainfluenzae. In patients with glomerular diseases, we examined renal-biopsy specimens for presence of bacterial antigen by immunofluorescence microscopy with rabbit antiserum against H parainfluenzae, and by enzyme-linked immunosorbent assay looked for IgA antibody against H parainfluenzae in patient sera. The rabbit antiserum recognised by immunoblotting four components of H parainfluenzae outer membranes (OMHP) of molecular weights 19.5, 30, 33, and 40.5 kDa. All 44 patients with IgA nephropathy and 2 of 39 patients with other glomerular diseases showed mesangial deposition of OMHP antigens (p < 0.001). Patients with IgA nephropathy had significantly more IgA antibody against H parainfluenzae than did patients with other glomerular diseases. IgA antibody in the sera of patients with IgA nephropathy recognised by immunoblotting the same four components of OMHP as recognised by rabbit antiserum. Glomerular deposition of OMHP antigens and the presence of IgA antibody against OMHP in patients with IgA nephropathy suggest that H parainfluenzae has a role in the aetiology of this disease.

Direct induction of tetraploids or homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydrostatic pressure.

Hydrostatic pressure and a dye plate method were used to investigate the direct induction of tetraploids or homozygous diploids from the industrial diploid or haploid yeast Saccharomyces cerevisiae. Above 200 MPa, hydrostatic pressure greatly inactivated the strains HF399s1 (alpha haploid), P-540 (a/alpha diploid), and P-544 (a/alpha diploid). At the same time, when pressure-treated cells of these strains were spread on a dye plate, some of the visible colonies were stained red/blue or dark blue (variant colonies); the rest stained violet, similar to colonies originating from diploid cells or haploid cells that were not pressure-treated. In addition, above 100 MPa, the formation of variant colonies increased with increasing pressure, and maximized (1 x 10(-1)) at 200 and 250 MPa, respectively. The size of almost all variant cells from P-544, P-540, and HF399s1 was visibly increased compared with that of untreated cells and the measured cellular DNA content of P-540 and HF399s1 was double that of untreated cells. Furthermore, based on random spore analysis and mass-matings, induced variants in the diploid strains were found to be tetraploid with an a/a/alpha/alpha genotype at the mating-type locus or, in the haploid strains, homozygous diploid with an alpha/alpha genotype. From these results we conclude that pressure treatment in combination with a dye plate is a useful method for strain improvement by direct induction of tetraploids or homozygous diploids from industrial strains whether diploid or haploid.

Intestinal fatty acid-binding protein as a sensitive marker of intestinal ischemia.

Determination of the serum level of intestinal fatty acid-binding protein has been used to detect rat intestinal ischemia following ligation or 30-min occlusion of the superior mesenteric artery. The normal values were under the minimal detectable level of less than 2 ng/ml in all the 10 rats. The serum fatty acid-binding protein level increased rapidly, to 340.7 +/- 54.6, 438.5 +/- 40.1, 388.1 +/- 37.4, and 292.2 +/- 95.7 ng/ml (P less than 0.01) at 1, 2, 4, and 8 hr after ligation, respectively. It also increased, to 347.2 +/- 127.7 ng/ml (P less than 0.01) at 1 hr, after a 30-min transient occlusion and then returned to a normal level. Histological studies showed destruction of the villi, disappearance of the mucosa, and transmural necrosis with the progress of time after ligation, while no remarkable morphological change was observed following 30-min transient occlusion. These observations strongly suggest that the intestinal fatty acid-binding protein is a useful biochemical marker for intestinal ischemia, particularly in the early reversible phase.

Mortality and histological findings of the brain during and after cerebral ischemia in male and female spontaneously hypertensive rats.

Mortality and pathological changes of the brain during and after cerebral ischemia induced by bilateral carotid artery occlusion (BCO) were studied in male and female spontaneously hypertensive rats (SHR). Systolic arterial blood pressure at rest was significantly higher in male SHR (228 +/- 13 mm Hg, mean +/- S.E.M.) than female (192 +/- 12) (P less than 0.05). The average survival time during permanent occlusion was 11 +/- 6 h (mean +/- S.D.) in male SHR and 17 +/- 7 in female (P less than 0.005), though the cumulative mortality during 24-h ischemia was not different between male (88%) and female SHR (84%). Severe ischemic changes of nerve cells in the brain, especially in the cortex and hippocampus, were observed in 50% of male SHR at 3-h ischemia, while only 15% was observed in female SHR even after 7-h ischemia. After the temporary ischemia followed by reperfusion for 24 h, the mortality was varied between male and female SHR; 0, 31 and 100% after 1-, 3- and 5-h ischemia, respectively, in male SHR and 0% after 1- to 3-h ischemia and 33% after 5- to 7-h ischemia, respectively, in female. Ischemic changes of the brain tissue, such as acidophilic cytoplasm, nuclear degeneration and intercellular edema, were more frequent and severe in male SHR than female after recirculation following 3- or 5-h ischemia. It is concluded that the mortality and post-ischemic viability seem to be determined by the duration of ischemia and also by the degree of the neuronal damage, and female SHR is more tolerated for ischemic insult in comparison to male SHR.

Effect of recirculation on the recovery of cerebral metabolism after experimental cerebral ischemia in male and female spontaneously hypertensive rats.

Lactate, pyruvate and adenosine triphosphate (ATP) concentrations in the brain were measured at the end of various periods of cerebral ischemia induced by bilateral carotid occlusion at 1-hour recirculation after the ischemia in spontaneously hypertensive rates (SHR). In both male and female SHR, a progressive and consistent increase in lactate and lactate/pyruvate ratio and a concomitant decrease in ATP were observed in the ischemic periods of 1, 3 or 5 h. Changes of these cerebral metabolites in females were two thirds to one half of those in males at corresponding periods of ischemia. At 1 h after recanalization of the occluded carotid arteries, metabolic derangements of the ischemic brain were little recovered in male SHR exposed to only 1-hour ischemia, whereas in female SHR the decreased ATP levels were recovered close to the nonischemic control level even after 7-hour ischemia. Furthermore, the increased lactate in female was attenuated to only one sixth of that in male at 1-hour recirculation after 5-hour ischemia. It is concluded that the recovery of the cerebral ischemic metabolism by reperfusion is better in female than male SHR, probably because of the smaller metabolic changes during the ischemic insult, and the fact that the degree as well as the duration of ischemia seem to be important factors for sufficient recovery from ischemic impairment of the brain.

Pulmonary hemorrhage in experimental cerebral ischemia in mongolian gerbils. Brain metabolism and lung pathology.

Pulmonary changes in acute cerebral ischemia were studied in anesthetized Mongolian gerbils, in which both carotid arteries were occluded simultaneously. Lactate, pyruvate and adenosine triphosphate (ATP) in the brain were measured as indicators of the severity of cerebral ischemia. Microscopic changes in the lung were arbitrarily scored from 0 (normal) to 3 points (severely affected) by the grade and the extent of lesions. Mean arterial pressure (MAP) was also measured through the cannulated femoral artery before and after carotid artery occlusion in a separate group of animals. Cerebral lactate was increased while ATP decreased in ischemic animals in which pulmonary changes such as intra-alveolar hemorrhages were prominent and frequent. The lung pathology score averaged 1.3 in animals with severe ischemia (lactate greater than or equal to 10 mM/Kg), 0.7 in moderate ischemia (5-10 mM/Kg) and 0.3 in mild or no ischemia (less than 5 mM/Kg), respectively, suggesting that severe brain ischemia may cause fulminant pulmonary changes. The mechanism of pulmonary lesions in acute cerebral ischemia is discussed.

Cerebrospinal fluid lactate and pyruvate concentrations in patients with malignant hypertension.

Lactate and pyruvate concentrations and acid-base parameters in cerebrospinal fluid (CSF) and arterial blood were measured in 21 patients with malignant hypertension ( MHT ), 19 with benign hypertension (BHT) and 21 normotensive subjects (NT). Average values for CSF lactate and lactate/pyruvate (L/P) ratio were significantly higher in MHT (1.90 +/- 10 mM/1, 19.2 +/- 1.0) than in either BHT (1.50 +/- 0.05 mM/l, 15.7 +/- 0.7) or NT (1.44 +/- 0.04 mM/1, 15.7 +/- 0.4). There was a linear correlation between CSF lactate and CSF pressure (r = 0.565, P less than 0.01), and the latter was also related to mean arterial pressure exceeding 150 mm Hg (r = 0.553, P less than 0.01). Such increases in the acid metabolites in CSF indicate that brain metabolism becomes anaerobic in MHT , probably due to increased intracranial pressure. Increased cerebrovascular permeability is also discussed as participating in causal mechanisms.

Sex difference of barbiturate-protection in experimental cerebral ischemia in spontaneously hypertensive rats (SHR).

Survival time following bilateral carotid occlusion was significantly longer in spontaneously hypertensive rats (SHR) anesthetized with barbiturate than in those with ether. Under barbiturate anesthesia, female SHR survived longer (16.6 h) than did males (10.9 h), whereas such sex difference was not found in those with ether anesthesia. The present results indicate sex difference of barbiturate-protection in cerebral ischemia.

[Effects of hypo- or hyperglycemia on brain metabolism in experimental cerebral ischemia].

Glucose, lactate, pyruvate and adenosine triphosphate (ATP) concentrations in the supratentorial brain tissue frozen in situ were measured one hour after bilateral carotid occlusion in spontaneously hypertensive rats, of which blood glucose levels were varied by intraperitoneally injected insulin (hypoglycemia), saline (normoglycemia) and 50% glucose (hyperglycemia). Cerebral glucose concentrations as well as blood glucose levels were significantly increased in hyperglycemic animals, and decreased in hypoglycemic ones. Cerebral lactate, and lactate/pyruvate ratio at one-hour ischemia tended to increase in hyperglycemic animals comparing with those in normoglycemic ones, although cerebral ATP levels were slightly higher in the former. In hypoglycemic animals with one-hour ischemia, cerebral lactate was less increased but ATP was significantly reduced. It has been reported that hyperglycemia has vulnerable effects on brain metabolism of complete cerebral ischemia, presumably due to hyperglycemia-induced lactic acidosis of the brain. In incomplete cerebral ischemia as demonstrated in the present study, however, ATP concentrations remained at slightly higher level, despite tendency to more increase in lactate in hyperglycemic animals, indicating that high blood glucose level might be beneficial, rather than vulnerable, to incomplete cerebral ischemia. On the other hand, hypoglycemia causes more severe impairment of the brain energy metabolism because of an insufficient supply of substrates to the brain.

Effects of hyperventilation on cerebral blood flow and brain tissue metabolism in normotensive and spontaneously hypertensive rats.

Cerebral vascular carbon dioxide (CO2) reactivities were compared in normotensive (NTR) and hypertensive (SHR) rats. Cerebral blood flow (CBF) in cortex and thalamus were evaluated before and during one hour of hyperventilation. After one hour of hyperventilation brain lactate, pyruvate, and ATP concentrations were also determined. Significant and similar reductions of CBF due to hyperventilation induce hypocapnia were found in both NTR and SHR groups. In contrast the percent increase in cerebrovascular resistance (CVR) per unit decrease in paCO2 was significant, indicating that hypocapnia induced vasoconstriction is greater in NTR than in SHR groups. During hyperventilation the average value for lactate in the NTR group was 3.98 mM/kg. In contrast it was 3.15 mM/kg in the SHR group, a significant difference (p less than 0.05). When paCO2 fell below 15 mm Hg the cerebral lactate increased strikingly in the NTR group and cortical CVR was reduced suggesting that an accumulation of the ischemic metabolites caused dilatation of the constricted cerebral vessels. In contrast the SHR group disclosed no such changes. The increase CVR characteristic of SHR appeared to diminish the cerebral vasoconstrictive response to hypocapnia. As a result ischemic metabolites in the brain do not increase in this group to the degree that they do in NTR.