Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells.

Growth factors hold great promise for regenerative therapies. However, their clinical use has been halted by poor efficacy and rapid clearance from tissue, necessitating the delivery of extremely high doses to achieve clinical effectiveness which has raised safety concerns. Thus, strategies to either enhance growth factor activity at low doses or to increase their residence time within target tissues are necessary for clinical success. In this study, we generated multivalent conjugates (MVCs) of basic fibroblast growth factor (bFGF), a key growth factor involved in angiogenesis and wound healing, to hyaluronic acid (HyA) polymer chains. Multivalent bFGF conjugates (mvbFGF) were fabricated with minimal non-specific interaction observed between bFGF and the HyA chain. The hydrodynamic radii of mvbFGF ranged from ∼50 to ∼75 nm for conjugation ratios of bFGF to HyA chains at low (10 : 1) and high (30 : 1) feed ratios, respectively. The mvbFGF demonstrated enhanced bioactivity compared to unconjugated bFGF in assays of cell proliferation and migration, processes critical to angiogenesis and tissue regeneration. The 30 : 1 mvbFGF outperformed the 10 : 1 conjugate, which could be due to either FGF receptor clustering or interference with receptor mediated internalization and signal deactivation. This study simultaneously investigated the role of both protein to polymer ratio and multivalent conjugate size on their bioactivity, and determined that increasing the protein-to-polymer ratio and conjugate size resulted in greater cell bioactivity.

Branching Analysis of Multivalent Conjugates Using Size Exclusion Chromatography-Multiangle Light Scattering.

Multivalent conjugates (MVCs) (conjugation of multiple proteins to a linear polymer chain) are powerful for improving the bioactivity and pharmacokinetics of a bioactive molecule. Since this effect is highly dependent upon the valency of the conjugated proteins, it is imperative to have a technique for analysis of the conjugation ratio. Studies of MVCs have used size exclusion chromatography-multiangle light scattering (SEC-MALS), which allows for the separate and individual analysis of the protein and biopolymer components based on their specific refractive index increment and UV extinction coefficient constants to determine the number of proteins bound per biopolymer molecule. In this work, we have applied traditional branching analysis to the SEC-MALS data, with the primary assumption that the polymer backbone can be used as the linear counterpart. We demonstrated good agreement between the branching values and the valency determined by traditional analysis, demonstrating that branching analysis can be used as an alternative technique to approximate the valency of MVCs. The branching analysis method also provides a more complete picture of the distribution of the measured values, provides important branching information about the molecules, and lowers the cost and complexity of the characterization. However, since MVC molecules are both conjugate molecules and branched molecules, the most powerful approach to their characterization would be to use both traditional multivalent conjugate analysis and branching analysis in conjunction.

sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo.

Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.

Multivalent hyaluronic acid bioconjugates improve sFlt-1 activity in vitro.

Anti-VEGF drugs that are used in conjunction with laser ablation to treat patients with diabetic retinopathy suffer from short half-lives in the vitreous of the eye resulting in the need for frequent intravitreal injections. To improve the intravitreal half-life of anti-VEGF drugs, such as the VEGF decoy receptor sFlt-1, we developed multivalent bioconjugates of sFlt-1 grafted to linear hyaluronic acid (HyA) chains termed mvsFlt. Using size exclusion chromatography with multiangle light scattering (SEC-MALS), SDS-PAGE, and dynamic light scattering (DLS), we characterized the mvsFlt with a focus on the molecular weight contribution of protein and HyA components to the overall bioconjugate size. We found that mvsFlt activity was independent of HyA conjugation using a sandwich ELISA and in vitro angiogenesis assays including cell survival, migration and tube formation. Using an in vitro model of the vitreous with crosslinked HyA gels, we demonstrated that larger mvsFlt bioconjugates showed slowed release and mobility in these hydrogels compared to low molecular weight mvsFlt and unconjugated sFlt-1. Finally, we used an enzyme specific to sFlt-1 to show that conjugation to HyA shields sFlt-1 from protein degradation. Taken together, our findings suggest that mvsFlt bioconjugates retain VEGF binding affinity, shield sFlt-1 from enzymatic degradation, and their movement in hydrogel networks (in vitro model of the vitreous) is controlled by both bioconjugate size and hydrogel network mesh size. These results suggest that a strategy of multivalent conjugation could substantially improve drug residence time in the eye and potentially improve therapeutics for the treatment of diabetic retinopathy.

A case in the spectrum of the oculo-encephalo-hepato-renal syndrome.

An 18-year-old male is presented with unprecedented central nervous system findings (cerebral dysplasia and sacral meningocele) possibly in the spectrum of the oculo-encephalo-hepato-renal syndrome. He had severe mental retardation, triplegia, epilepsy, retinitis pigmentosa, and chronic renal failure. Magnetic resonance imaging demonstrated cerebral dysplasia (left dominant abnormal gyri, hypoplastic white matter, basal ganglia, and thalamus, and absence of the septum pellucidum) and the hypoplastic cerebellum and brainstem. A sacral meningocele was observed first at 16 years of age. His renal function gradually worsened after 11 years of age. His liver function was normal. The previously reported 72 cases with the oculo-encephalo-hepato-renal syndrome are reviewed.

The efficiency of performing ultrasound-guided fine-needle aspiration biopsy following mass screening for thyroid tumors to avoid unnecessary surgery.

Thyroid masses are a common clinical finding, and their management remains controversial. The purpose of this study was to evaluate the clinical effect of performing routine ultrasound (US) examinations and US-guided fine-needle aspiration biopsy (US-FNAB) in the management of diffuse or nodular goiter diagnosed by mass screening. Mass screening carried out from 1993 to 1996 revealed 444 women with goiter, 322 of whom had diffuse goiter and 122 had nodular goiter. All of these patients underwent US examination, the results of which determined that 169 should undergo US-FNAB to confirm an accurate diagnosis of their thyroid tumors. Histological examinations after surgical resection revealed that 12 of the 322 patients with diffuse goiter (3.7%) and 23 of the 122 with nodular goiter (18.9%) had malignant tumors. Among the 61 thyroid tumors surgically verified, US-FNAB yielded a sensitivity rate of 93%, a specificity rate of 81%, and an accuracy rate of 90%. Insufficient aspiration was obtained from 5%. Performing US-FNAB-resulted in an elevation in the percentage of malignant tumors yielded at surgery of up to 72%. Thus, ultrasonography followed by US-guided-FNAB could be a useful routine method of evaluating thyroid tumors detected by mass screening. Moreover, a greater number of unnecessary thyroid operations can be avoided by performing US-FNAB rather than FNAB alone.

Excitability recovery curves of blink reflexes in patients with athetotic cerebral palsy.

We evaluated the brainstem function or its excitability by the blink reflex evoked with the electrical stimulation to the supraorbital nerve in 10 patients with athetotic cerebral palsy compared with 10 normal subjects and 7 spastic type patients. There were no differences in stimulus intensity, latency of R1 and R2 components, and duration and area of EMG activity of the R2 component of the blink reflex elicited by single stimulation among the two patients' groups and normal subjects. R1 recovery cycle to paired stimuli in the athetotic group showed a facilitation of the test responses by the conditioning stimuli at 100 and 200 ms intervals, but were not significantly different from those in the normals. On the other hand, the R2 recovery curve in the athetotic group showed a significant hyperexcitability at all intervals from 100 to 600 ms compared to the normals. Our results from the R2 hyperexcitable recovery to paired stimuli are indicative of increased brainstem interneuronal excitability in athetotic patients and similar to the results reported in the disorders of the basal ganglia, i.e. Parkinson's disease, dystonia and blepharospasm. We suggest that this hyperexcitability might be caused by abnormal input possibly from the basal ganglia upon these brainstem interneurons.

Central motor reorganization in cerebral palsy patients with bilateral cerebral lesions.

Transcranial magnetic stimulation (TMS) has been used to describe cortical plasticity after unilateral cerebral lesions. The objective of this study was to find out whether cortical plasticity occurs after bilateral cerebral lesions. We investigated central motor reorganization for the arm and leg muscles in cerebral palsy (CP) patients with bilateral cerebral lesions using TMS. Seventeen patients (12 with spastic diplegia, 1 with spastic hemiplegia, and 4 with athetoid CP) and 10 normal subjects, were studied. On CT/MRI, bilateral periventricular leukomalacia was observed in all spastic patients with preterm birth. In two normal subjects, motor responses were induced in the ipsilateral tibialis anterior, but no responses were induced in any normal subject in the ipsilateral abductor pollicis brevis (APB) or biceps brachii (BB). Ipsilateral responses were more common among CP patients, especially in TMS of the less damaged hemisphere in patients with marked asymmetries in brain damage: in 3 abductor pollicis brevis, in 6 BBs, and in 15 tibialis anteriors. The cortical mapping of the sites of highest excitability demonstrated that the abductor pollicis brevis and BB sites in CP patients were nearly identical to those of the normal subjects. In patients with spastic CP born prematurely, a significant lateral shift was found for the excitability sites for the tibialis anterior. No similar lateral shift was observed in the other CP patients. These findings suggest that ipsilateral motor pathways are reinforced in both spastic and athetoid CP patients, and that a lateral shift of the motor cortical area for the leg muscle may occur in spastic CP patients with preterm birth.

The suppression of postoperative liver metastasis caused by the continuous intraportal infusion of angiogenesis inhibitor FR-118487 in a rabbit colon cancer model.

The inhibitory effect of FR-118487, a potent angiogenesis inhibitor, on neovascularization induced by the VX2 tumor was confirmed in a rabbit corneal assay. The antimetastatic effect of FR-118487 was also investigated in 21 rabbits. Spontaneous liver metastases were induced by VX2 tumor cell implantation into the ascending colonic wall. FR-118487 was then infused continuously into the portal vein for 7 days after resection of the primary lesion at a dose of 1 mg/kg/day (FR-1 group) or 3 mg/kg/day (FR-3 group). The incidence of liver metastases was 71.4%, occurring in 5 of 7 rabbits, in each of the FR-1 and FR-3 groups, compared with 100%, being all of 7 rabbits, in the control group. The number of metastatic foci tended to be less in the FR-1 (31.0 +/- 36.0) and FR-3 (24.6 +/- 45.1) groups than in the control group (83.7 +/- 73.9) and the weight of metastatic foci was significantly less in the FR-1 (1.4 +/- 1.8 g) and FR-3 (1.3 +/- 2.0 g) groups than in the control group (6.5 +/- 4.9 g) (P < 0.05). However, leakage of the colonic anastomosis and body weight loss were limited to the FR-3 group. These results suggest that the continuous intraportal infusion of FR-118487 at 1 mg/kg/day suppressed liver metastases by inhibiting angiogenesis, without producing any adverse effects.

[The blink reflex in severely handicapped patients with dysphagia].

We investigated the blink reflex in 27 severely handicapped patients. 15 males and 12 females, from 5 to 60 years old. They were divided into three groups; the tube-feeding group (9 patients), the oral-feeding group (9 patients), and the mixed-feeding group (9 patients). Seven normal subjects also were included in this study. There was a significant difference in the late ipsilateral component (R2) between the tube-feeding and oral-feeding groups. The average R 2 time were 43.0 msec. in the tube-feeding group and 36.7 msec. in the oral feeding group. The auditory brainstem response (ABR) showed no significant difference between the two groups. These results suggested that the swallowing function in the patients needed for tube feeding was affected by not only the cortical lesions but also the brainstem ones. Furthermore, it is speculated that the degree of brainstem dysfunction in the tube-feeding group was more severe than in the non-tube-feeding group. The blink reflex is one of the useful electrophysiological parameters for evaluating the brainstem function in severely handicapped patients with dysphagia.

[Suppression of postoperative liver metastases from colon cancer by continuous intraportal infusion of an angiogenesis inhibitor FR-118487].

A rabbit VX2 colon cancer model with spontaneous liver metastases was used to evaluate the antitumor effect of an angiogenesis inhibitor, FR-118487. FR-118487 (1 mg/kg/day) was infused continuously into the portal vein for a week after resection of primary colon cancer lesions (FR group). The incidence of liver metastases was 71.4% (5/7) in FR group, and 100% (7/7) in control group. The number and the weight of liver metastatic nodules were 31.0 +/0 36.0 and 1.4 +/- 1.8 g in FR group versus 83.7 +/- 73.9 and 6.5 +/- 4.9 g in control group, respectively. The metastases in FR group were significantly decreased in weight compared with those in control group (p < 0.05). No anastomotic leakage was recognized in either group. No side effects of FR-118487 such as body weight loss were found. Continuous intraportal infusion of FR-118487 in the early postoperative period may be effective to suppress liver metastases from colon cancer by inhibiting the angiogenesis concerning liver metastases.

[Late progression of pulmonary hypertension in a patient operated for patent ductus arteriosus: pulmonary angiopathy or thromboembolism?].

A 16-year-old girl developed dyspnea 9 years after surgery for patent ductus arteriosus with pulmonary hypertension (pulmonary to systemic peak systolic pressure ratio; Pp/Ps 1.0). The postoperative course had been uneventful with a decreased Pp/Ps. It was revealed this time, however, that the pulmonary arterial pressure again elevated to be 186/133 mmHg (Pp/Ps 1.90). Postoperative progression of pulmonary angiopathy was first suspected. Whereas, the findings with pulmonary perfusion scintigraphy and arteriography were remarkably different between the right and left lungs, suggesting that the progression of pulmonary hypertension was not caused by that of pulmonary angiopathy but by the association of thromboembolism in small pulmonary arteries. The patient succumbed to intractable cardiopulmonary failure, strongly appealing for the necessity of organ-transplantation therapy in Japan. This case also indicated the particular importance of a close follow-up of the cases with pulmonary hypertension remaining after corrective surgery.

Quantitative histologic study of sural nerves in xeroderma pigmentosum.

The sural nerves of 2 siblings, 7 and 6 years of age with Group A xeroderma pigmentosum, were biopsied. The densities of myelinated fibers, 5,808/mm2 and 5,163/mm2, respectively, were strikingly decreased in comparison to control data. Both large and small myelinated fibers were reduced. Electron microscopy demonstrated many collagen fibers in the endoneurium and some collagen pockets. The loss of myelinated fibers was less severe than in previously reported patients. This discrepancy may be due to age differences at biopsy; our patients were biopsied at the ages of 7 and 6 years, while those patients reported previously were 10 years of age or older. The incidence of neurologic manifestations in xeroderma pigmentosum may increase after 6 years of age.

Lipid composition of the cerebral gray and white matter in a case with Fukuyama type congenital muscular dystrophy.

When compared with an age-matched normal control and a Duchenne muscular dystrophy (DMD) case, the patient with FCMD had an unusual ganglioside pattern in the cerebral gray matter. The total level of lipid-bound sialic acid in the cerebral gray matter was also slightly decreased. However, other lipid compositions of the cerebral gray and white matter were normal compared with those in the control case.

Computed tomography in patients with Ehlers-Danlos syndrome.

Three patients with Ehlers-Danlos syndrome were reported. Unusual findings on computed tomography were seen in two of the three patients. One case showed peculiar and marked dilatation of the 4th ventricle, supracerebellar cistern and lateral ventricle. The other case presented disproportionate enlargement of the anterior horn of the lateral ventricle. These CT findings in the two patients suggest that developmental abnormalities may constitute a structural defect.

Intracranial hemorrhage in the full-term neonate and young infant: correlation of the location and outcome.

The cranial computed tomography (CT) and outcome for 13 full-term neonates and 12 young infants with intracranial hemorrhage (ICH) were studied. The full-term neonates had perinatal asphyxia or neurological signs such as seizures. All infants were breast-fed and showed bleeding diathesis. In the full-term neonates there was a high incidence of intraventricular hemorrhage (IVH) and hemorrhage around the falx. The location of the hemorrhage on CT and brain pathology suggested that the original site of IVH might be the choroid plexus vessels in the lateral ventricle or in the subependymal layer. On the other hand, the sites of ICH in infants were multifocal compared with those in full-term neonates. Subdural hemorrhage (SDH) was seen more frequently and IVH less frequently in infants than in full-term neonates. The cases with SDH frequently showed accompanying cerebral infarction followed by porencephaly. Thus, SDH with cerebral low density on CT may predict a poor prognosis.