Structure of the Plexin Ectodomain Bound by Semaphorin-Mimicking Antibodies.

Semaphorin family proteins act on cells to mediate both repulsive and attractive guidance via binding to plexin family receptors, thereby playing fundamental roles in the morphogenesis and homeostasis of various tissues. Although semaphorin-plexin signaling is implicated in various diseases and is thus a target of intensive research, our mechanistic understanding of how semaphorins activate plexins on the cell surface is limited. Here, we describe unique anti-plexin-A1 antibodies that can induce a collapsed morphology in mouse dendritic cells as efficiently as the semaphorin 3A (Sema3A) ligand. Precise epitope analysis indicates that these "semaphorin-mimicking" antibodies dimerize cell-surface plexin-A1 by binding to the N-terminal sema domain of the plexin at sites away from the interface used by the Sema3A ligand. Structural analysis of plexin-A1 fragments using negative stain electron microscopy further revealed that this agonistic capacity is closely linked to the location and orientation of antibody binding. In addition, the full-length plexin-A1 ectodomain exhibited a highly curved "C" shape, reinforcing the very unusual dimeric receptor conformation of this protein at the cell surface when engaged with Sema3A or agonistic antibodies.

Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity.

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.

A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model.

Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.

Intravenously administered darbepoetin alfa once a week could maintain hemoglobin level more efficiently than once every 2 weeks in patients on hemodialysis.

BACKGROUND: Darbepoetin alfa, which has a much longer half-life than recombinant human erythropoietin, is used to treat renal anemia. However, there are few reports investigating the efficacy of darbepoetin alfa administered every 2 weeks (Q2W). METHODS: We performed the single-center, prospective, and randomized study in chronic hemodialysis patients. Clinically stable patients on hemodialysis were recruited, and darbepoetin alfa 15-40 microg/week was administered intravenously once a week (QW) to achieve a hemoglobin (Hb) level of 10.5-12.0 g/dl for 8 weeks prior to randomization. The patients were randomly assigned to 2 different dosing frequency groups: once a week (QW) or every 2 weeks (Q2W). We switched to a double dose in the Q2W group. We measured Hb level every 2 weeks and administered darbepoetin alfa to achieve an Hb level of 10.5-11.5 g/dl. The primary endpoints were the weekly dose of darbepoetin alfa administered at week 24. RESULTS: We assigned 19 and 20 patients into QW and Q2W, respectively. There were no significant differences between the groups in Hb, transferrin saturation, ferritin, and weekly dose of darbepoetin alfa at end of the baseline period. There was no significant difference in Hb level at week 24, at which time the weekly dose requirement and dose per dry body weight were much higher in the Q2W than in the QW group. CONCLUSION: Administration of darbepoetin alfa Q2W could maintain Hb level similarly to to that obtained QW, but we did not confirm efficiency at a higher dose requirement or blood pressure elevation.

Relationship between serum 1,25-dihydroxyvitamin D and mortality in patients with pre-dialysis chronic kidney disease.

BACKGROUND: It is known that vitamin D has many functions besides involvement in calcium metabolism. It has recently been recognized that vitamin D deficiency is associated with mortality, especially in cardiovascular disease (CVD). Vitamin D deficiency is common in end-stage renal disease, but develops from the early stage of chronic kidney disease (CKD). So we investigated whether the serum level of the activated form of vitamin D (1,25-dihydroxyvitamin D) affected mortality in patients with CKD stages 3 and 4. METHODS: Between January 1, 1995, and June 30, 2006 we measured serum 1,25-dihydroxyvitamin D In 226 patients with CKD stages 3 and 4 and classified the results into two groups depending on whether the level was below (group I) or above (group II) 20 pg/ml. We ended the follow-up period on December 31, 2006. We compared all-cause and cardiovascular mortality between the two groups. We also examined predictors of mortality by using Cox proportional regression analysis. RESULTS: Two-hundred and twenty-six patients (67 men and 159 women, mean age 67.0) were registered in this study, and groups 1 and 2 comprised 84 and 142 patients, respectively. During the follow-up period 43 patients died. CVD was the major cause of death, followed by infectious disease. The Kaplan-Meier survival curve revealed that all-cause mortality was significantly higher in group I, but a significant difference between CVD mortality in the two groups was not demonstrated. By Cox proportional regression analysis, group I was related to all-cause mortality, but this was not proved to be an independent predictor. CONCLUSION: The results suggested that serum level of 1,25-dihydroxyvitamin D was associated with all-cause mortality in patients with CKD stages 3 and 4.

Relationship between renal function at the time of percutaneous coronary intervention and prognosis in ischemic heart disease patients.

BACKGROUND: As hypertension and diabetes mellitus increase, the number of patients developing complications of cardiovascular disease (CVD) associated with conventional risk factors is increasing. In addition to these risk factors for CVD, chronic kidney disease (CKD) has also been reported to play an important role. We investigated the association of representative ischemic heart disease and CKD. METHODS: Between July 1, 2000, and June 30, 2005, a total of 790 patients who underwent percutaneous coronary intervention (PCI) for angina pectoris or myocardial infarction in our division of cardiovascular disease were reviewed. Serum markers at the implementation of PCI were compared in patients classified according to renal function. For prognosis, in-hospital mortality, 1-year survival rate, overall mortality, and CVD death were investigated. Changes in renal function were also monitored during the follow-up period. The glomerular filtration rate (GFR) was calculated by the Modification of Diet in Renal Disease Study Group (MDRD) formula. RESULTS: The mean estimated GFR (eGFR) at PCI implementation was 66.2 +/- 21.0 ml/min/1.73 m(2). Stage 2 CKD was shown in 51.5% of all the patients. During the overall follow-up period, 126 patients died. With the progression of CKD stage, all-cause, CVD, and in-hospital mortality increased, and the 1-year survival rate decreased. It was proved that a medical history of hypertension, hyperlipidemia, and diabetes, multiple vessel lesions, hypoalbuminemia, C-reactive protein (CRP), and estimated GFR were independent risk factors for all-cause death. In CVD death, in addition to the above risk factors, anemia and total cholesterol were also an independent risk factor. Renal function deteriorated significantly during the follow-up period. CONCLUSIONS: Patients with ischemic heart disease requiring PCI often develop renal dysfunction, which may considerably affect prognosis.

Effect of an educational program on the predialysis period for patients with chronic renal failure.

BACKGROUND: The purpose of the treatment and management of chronic renal failure during the predialysis period is mainly to retard the progression of the deterioration of renal function. Optimal dialysis initiation is important to improve the patient's outcome after therapy. We investigated whether providing information through an original educational program could facilitate dialysis initiation, with the patient in a better condition, and therefore greater cost-effectiveness. METHODS: One hundred and seventy-six patients who underwent dialysis initiation for chronic renal failure in our hospital between April 2002 and March 2005 were divided into two groups according to their participation or nonparticipation in an educational program. Participation in the education program was of their own free will. The instructors consisted of nephrologists, nursing staff, clinical engineering technologists, national registered dietitians, and medical social workers. We investigated whether the education program facilitated trouble-free dialysis initiation by comparing findings of blood tests at the start, the existence of heart-failure symptoms, type of blood access, percentage of scheduled initiation, and the number of days and cost of hospitalization as indices between participating and nonparticipating groups. RESULTS: The number of patients using a double-lumen dialysis catheter, and the duration and cost of hospitalization in training the participating group, were significantly less than those in the nonparticipating group. Although there was no significant difference in renal function at the initiation of renal replacement therapy (RRT) between the two groups, serum albumin, hemoglobin, and hematocrit in the participating group were significantly higher than those of the nonparticipating group. CONCLUSIONS: This study suggests that providing sufficient information before dialysis initiation may be effective in both physical condition at dialysis initiation, and medical economic benefits through the understanding of the dialysis.

A model of spontaneous periodontitis in the miniature goat.

BACKGROUND: The pathogenesis and progression of periodontal disease have been extensively studied through the use of animal models. However, no animal model has yet been established that is precisely similar to periodontitis in humans. In the present study, we examined the use of Shiba goats as a model for spontaneous periodontitis. METHODS: Thirty-four Shiba goats (seven males and 27 females, aged 10 to 98 months) were used. We examined periodontitis in Shiba goats clinically, histopathologically, and microbiologically. RESULTS: The mean probing depth (PD) of the 68 teeth examined was 2.7 +/- 0.8 mm. The incidences of PD > or =3 and 4 mm were 46.4% and 22.1%, respectively. The incidence of bleeding on probing in 68 sites and in 34 animals was 60.7% and 73.5%, respectively. The formation of vertical alveolar bone defects and downgrowth of gingival epithelial cells were found in the areas of periodontitis. The prevalence of Tannerella forsythensis, Campylobacter rectus, Fusobacterium necrophorum, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans in subgingival plaque by polymerase chain reaction was 46.4%, 28.5%, 28.5%, 17.8%, and 3.5%, respectively. These percentages were increased in subgingival plaque from PD > or =3 mm. CONCLUSIONS: The clinical, histopathological, and microbiological features of spontaneous periodontitis in Shiba goats were somewhat similar to those in human periodontitis. Moreover, there are some advantages of using the Shiba goat; the size of the oral cavity is suitable for periodontal treatment, and handling and housing are relatively easy. Therefore, these results suggest that the Shiba goat is a useful animal model for human periodontitis.

[Effect of vitamin D on remaining kidney function after chronic kidney failure in preservation period patient].

It is necessary to manage the secondary hyperparathyroidism at the early stage. We concern about vitamin D, one of the treatment for secondary hyperparathyroidism has some adverse effects on the kidney function. We administered oral vitamin D (alfa calcidol 0.25 microg/day) of 21 outpatients who have chronic renal failure of preservation period and observed clinical laboratory test result of serum creatinine, adjusted calcium, intact phosphate. Renal function is evaluated by the value of the decline of serum creatinine reciprocal. A small dosage of oral vitamin D may not effect on kidney function, but we should examine this though studying more cases.

Combined effects of Nd:YAG laser irradiation with local antibiotic application into periodontal pockets.

The purpose of the present study was to assess the effects of Nd:YAG laser irradiation into periodontal pockets with or without the combination of local antibiotic application on clinical parameters and microbiological prevalence. Sixteen patients, each of whom had 4 or more sites with probing depth e x 4mm were included in this study. They were monitored clinically and microbiologically at baseline, 1 and 3 months after the treatment. Subgingival plaque samples were taken from periodontally involved sites with a probing depth of e x 4mm. A total of 135 sites were randomly assigned to the following treatments; Nd:YAG laser alone (Group L: 10 pps, 200 mJ for 90 sec, n = 37), local minocycline administration following laser treatment (Group LP, n = 33), povidone-iodine irrigation following laser treatment (Group LI, n = 33), and control group (Group C: sham procedure, n = 32). The prevalence of 18 subgingival taxa were determined using the checkerboard technique. The mean value of the pocket probing depth (PPD) significantly decreased in Group L, Group LP and Group LI, and the mean clinical attachment loss (CAL) decreased in all three treatment groups. Multivariate logistic regression analysis showed that Group LP showed the most improvement in PPD or CAL at 3 months. The proportions of Porphyromonas gingivalis, Tannerella forsythia (formerly Bacteroides forsythus) and Prevotella intermedia were significantly lower in Group LP than in Group L after 3 months. These results showed that Nd:YAG laser irradiation plus local minocycline provides a much greater reduction in PPD, CAL and the amount of periodontopathogenic bacteria than laser irradiation alone in periodontitis patients.