An Effective New Cryopreservation Procedure for Pancreatic Islets Using Hollow Fiber Vitrification.

The present study aimed at establishing a new cryopreservation method for mouse pancreatic islets by vitrification using hollow fibers as a container. A unique feature of the hollow fiber vitrification (HFV) method is that this method achieves stable vitrification using a minimum volume of cryoprotectant (CPA) solution, thereby ensuring high viability of the islets. The cytotoxicity, optimum composition, and concentration of the CPAs for vitrifying islets were examined. The viability, functional-integrity of vitrified islets were evaluated in comparison with those vitrified by conventional methods. Insulin secretion was measured in vitro by a static incubation assay and the metabolic functions was tested after transplantation into Streptozotocin-induced diabetic mice. The combination of 15% dimethyl sulfoxide+15% ethylene glycol resulted in the best CPA solution for the HFV of islets. HFV showed the highest viability in comparison to 2 vitrification methods, open pulled straws and vitrification with EDT324 solution. The vitrified islets stably expressed ß-cells markers NeuroD, Pancreatic and duodenal homeobox-1, and MafA. Transplantation of the vitrified islets achieved euglycemia of the host diabetic mice and response to an intraperitoneal glucose tolerance test to a similar extent as non-vitrified transplanted islets. The HFV method allows for efficient long-term cryopreservation of islets.

Diffusion-weighted imaging for predicting new compression fractures following percutaneous vertebroplasty.

BACKGROUND: Percutaneous vertebroplasty (PVP) is a technique that structurally stabilizes a fractured vertebral body. However, some patients return to the hospital due to recurrent back pain following PVP, and such pain is sometimes caused by new compression fractures. PURPOSE: To investigate whether the apparent diffusion coefficient (ADC) of adjacent vertebral bodies as assessed by diffusion-weighted imaging before PVP could predict the onset of new compression fractures following PVP. MATERIAL AND METHODS: 25 patients with osteoporotic compression fractures who underwent PVP were enrolled in this study. ADC was measured for 49 vertebral bodies immediately above and below each vertebral body injected with bone cement before and after PVP. By measuring ADC for each adjacent vertebral body, ADC was compared between vertebral bodies with a new compression fracture within 1 month and those without new compression fractures. In addition, the mean ADC of adjacent vertebral bodies per patient was calculated. RESULTS: Mean preoperative ADC for the six adjacent vertebral bodies with new compression fractures was 0.55 x 10(-3) mm(2)/s (range 0.36-1.01 x 10(-3) mm(2)/s), and for the 43 adjacent vertebral bodies without new compression fractures 0.20 x 10(-3) mm(2)/s (range 0-0.98 x 10(-3) mm(2)/s) (P < 0.001). Mean preoperative ADC for the six patients with new compression fractures was 0.55 x 10(-3) mm(2)/s (range 0.21-1.01 x 10(-3) mm(2)/s), and that for the 19 patients without new compression fractures 0.17 x 10(-3) mm(2)/s (range 0.01-0.43 x 10(-3) mm(2)/s) (P < 0.001). CONCLUSION: The ADC of adjacent vertebral bodies as assessed by diffusion-weighted imaging before PVP might be one of the predictors for new compression fractures following PVP.

Seizures with apnea in children.

Five children who had apneic attacks as a manifestation of epileptic seizures are reported. In three children, the apneic attacks were the sole symptom of epileptic fits. The other two children had additional types of seizures. The apneic seizures usually lasted one to two minutes and were characterized by respiratory embarrassment rather than complete arrest. These seizures are usually accompanied by cyanosis which appears early during the course of the attack. Although consciousness seemed to be impaired, this was difficult to assess accurately in infants. The ictal EEGs disclosed paroxysmal discharges of focal onset in all cases, consisting of high amplitude theta-waves, gradually mixed with or replaced by sharp waves or spikes in some and fast waves followed by slower waves in others. These occurred in temporal areas in three patients and in frontal and central areas in one patient each. Interictal EEGs, however, revealed paroxysmal discharges in only one patient. Apneic attacks in these infants are considered to be an integral part of the manifestations of complex partial seizures originating mostly in the limbic system.

Detection of platelet mitochondrial DNA deletions in Kearns-Sayre syndrome.

To establish a noninvasive genetic diagnosing method for Kearns-Sayre syndrome, the authors used the polymerase chain reaction (PCR) technique for detecting mitochondrial DNA (mtDNA) deletions in the platelets and directly sequenced the crossover regions of the deleted mtDNA using the fluorescence-based automated sequencing system. The mtDNA deletions were identified in the platelets of three of four patients. The sizes and locations of deletions were determined by the nesting primer PCR method, in which the primary PCR products derived from deleted mtDNAs undergo reamplification using a series of nesting primers. With the fluorescence-based sequencing of templates amplified by the asymmetric PCR method, deleted mtDNA was sequenced directly without cloning. In patient 1, guanine (G) was found at the boundaries of a deleted segment spanning 8400 base pairs (bp) between the CO1 and ND6 genes. In patient 2, a 9-bp directly repeated sequence of 5'-ACCTCCCTC-3' (where A = adenine, C = cytosine, and T = thymine) was found at the boundaries of a deleted segment spanning 7221 bp between the CO1 and ND5 genes. In patient 3, an 8-bp sequence of 5'-TCGCTGTC-3' was found at the boundaries of a deleted segment spanning 4664 bp between the ATPase6 and ND5 genes. Deletions were not detected in the mtDNA of patient 4 or in that of the mothers of the patients. Previously, the genetic diagnosis of this syndrome required muscle biopsy specimens and the use of Southern blot analysis. However, this method requires neither muscle biopsy nor isotopes and is more rapid than the Southern blot method.(ABSTRACT TRUNCATED AT 250 WORDS)

Lectin-histochemical reactivity of sialic acid in breast cancer and its relationship to prognosis using limulus polyphemus agglutinin.

Studies of circulating sialic acid have revealed its relationship with a variety of malignant tumors. It is not vet clear whether sialic acid could be used as a prognostic marker of breast cancer, and few studies have examined sialic acid expression in the cell membrane and cytoplasm of breast cancer cells by means of the lectin-histochemical technique. In the present study, we used biotinylated limulus polyphemus agglutinin (LPA), a special binding lectin of sialic acid, to stain sialic acid in breast cancer cells. Of the 104 cases of breast cancer examined, 59 (56.7%) positive cases were observed. There was a significant correlation between the LPA staining and the clinicopathologic features of all patients, including pathological stage and lymph node metastasis. Among the 100 patients who underwent curative operation, the mean disease-free survival rate of the 45 patients who were LPA-negative was significantly higher than that of the 55 LPA-positive patients (p<0.05). These results suggest that the positive expression of sialic acid in breast cancer could be used as a marker of malignancy potential, as well as a poor survival factor, and the biotinylated LPA assay may provide a convenient and useful method to predict the prognosis of breast cancer.

Bcl-2 protein expression in breast cancer and its relationship to prognosis.

The expression of bcl-2 protein was studied in invasive breast cancer by immunohistochemistry. Fourty-six (56.8%) bcl-2 protein-positive tumors were found in 81 breast cancers. There was no significant correlation between the bcl-2 protein immunoreactivity and histologic type, primary tumor status, or lymph node metastasis. However, a strong positive relationship was demonstrated between bcl-2 immunoreactivity and estrogen receptor status. The 5-year survival rate and disease-free survival rates were 73.7% and 71.1% of patients with bcl-2-positive tumors, and 62.5% and 58.0% of those with bcl-2-negative tumors; these differences between the two groups of patients wete significant (p<0.05). In multivariate analysis using Cox regression model, bcl-2 immunoreactivity emerged as an independent prognostic indicator in breast cancer patients.

Lateral gap junction connections between retinal amacrine cells summating sustained responses.

Vertebrate retinal amacrine cells produce transient or sustained responses. Sustained depolarizing amacrine cells in the dace retina were identified by their intracellularly recorded responses to light flashes. The response amplitude produced a notable spatial summation which exceeded that of individual dendritic arbors. When sustained type amacrine cells were intracellularly injected with Lucifer Yellow and biocytin, there was extensive transfer of biocytin, but not Lucifer Yellow, to surrounding cells with similar cellular morphology. Ultrastructural analysis of the interconnections by electron microscopy revealed the presence of gap junctions at the contact area, which did not include conventional synapses. Present results demonstrate that sustained response amacrine cells make direct electrical connections between the cells of the same type and electrical coupling may contribute to extension of their receptive fields.

Concomitant administration of sodium dichloroacetate and thiamine in west syndrome caused by thiamine-responsive pyruvate dehydrogenase complex deficiency.

We treated a female patient with West syndrome caused by thiamine-responsive pyruvate dehydrogenase complex (PDHC) deficiency. Infantile spasms occurred in association with elevated blood and CSF lactate concentrations; these symptoms disappeared when lactate concentrations had been lowered by treatment with concomitant sodium dichloroacetate (DCA) and high dose thiamine. Sequencing the patient's PDHC E(1)alpha subunit revealed a substitution of serine for glycine at position 89 in exon 3 (G89S). This mutation must be a de novo mutation because it was not found in either parents' genome DNA. To our knowledge, five previously described patients with PDHC deficiency have displayed the West syndrome. All six known patients, including our own, were female, even though an approximately equal number of males and females have been identified with PDHC deficiency and overall West syndrome occurs somewhat more frequently in males. These results indicated that West syndrome occurred more frequently in female patients with PDHC deficiency. It is suggested that lactate concentration should be measured in patients with West syndrome for potential PDHC deficiency, especially in females.

A simple fluorometric method for the determination of serum free carnitine.

A new fluorometric method for the determination of serum free carnitine is described. The addition of carnitine to a system containing carnitine acetyltransferase (EC 2.3.1.7) and acetyl-CoA gives rise to the formation of CoA. The system is coupled to N-(p-(2-benzimidazolyl)-phenyl)-maleimide (BIPM). A fluorescent agent, CoA-BIPM, is produced proportionally to concentration of carnitine. By measuring the fluorescence intensity of BIPM, the carnitine content of serum can be determined. The coefficients of variation, within-run and between-run, of the method were 5.2 and 2.6%, respectively. Recovery of carnitine added to serum was 98-113%. Comparison with a colorimetric method showed a good correlation (r greater than 0.90). The method has sufficient sensitivity to measure concentrations as low as 10 mumol/l.

Benign infantile epilepsy with complex partial seizures.

Benign infantile epilepsy with complex partial seizures is characterized by a high incidence of family history of benign childhood convulsions, normal development prior to onset, infantile onset, no underlying disorders, no neurological abnormalities, normal interictal EEGs, good response to treatment, and complete remission with normal developmental outcome. Seizures often occur in clusters, consisting of motion arrest, decreased responsiveness, staring or blank eyes mostly with simple automatisms, and mild convulsive movements associated with focal paroxysmal discharges, most frequently in the temporal area.

Significance of tumor marker SLX in colorectal cancer.

We examined serum SLX for its significance as a tumor marker in 109 colorectal cancer patients. There a close correlation with immunohistochemical expression of SLX and serum SLX level. Serum SLX was positive in 16.5% of 109 patients with colorectal cancers. There was no significant correlation between serum SLX level and histologic type or primary tumor status. There were significant correlations between serum SLX positive rates and both lymph node and hematogenous metastasis. In 7 SLX positive cases who underwent curative resection, 4 patients had already recurrence in the liver. Our findings suggest that serum SLX values may be a biologic marker of metastasis.

Sialyl-lewis-x antigen immunoreaction of colorectal-cancer and its relationship to hematogenous metastasis.

The adhesion of cancer cells to endothelial cells of the target organ is one of the most important steps of hematogenous metastasis. Especially, sialyl Le(X) plays an important role in defining the metastasis. The expression of sialyl Le(X) antigen in colorectal cancer and its usefulness not only as an indicator of metastatic potential but also as a prognostic factor was studied immunohistochemically. Fifty-five (32.4%) sialyl Le(X) antigen-positive tumors were found in 170 colorectal cancers. There was a significant correlation between the expression of sialyl Le(X) antigen and the histological tumor type, venous invasion, lymph node metastasis, as well as liver metastasis. Hematogenous metastases were significantly more frequent in patients with sialyl Le(X)-positive tumor than in those with sialyl Le(X)-negative tumor; and prognosis was significantly poorer in the former. The results suggest that sialyl Le(X) antigen plays a role in hematogenous metastasis of colorectal cancer, and that the expression of sialyl Le(X) is associated with poor prognosis.

Serial changes of cranial computerized tomographic findings in Wilson disease during D-penicillamine therapy.

Serial changes of cranial CT findings were studied in three siblings with Wilson disease during the course of D-penicillamine therapy. The older two cases with neurological presentation revealed low density areas in the region of the basal ganglia on the CT scans performed before treatment. Mild cortical atrophy and mild enlargement of the lateral and the third ventricles were also observed. The chelating therapy resulted in a considerable improvement of neurological symptoms and disappearance of Kayser-Fleischer rings. The low density areas of the basal ganglia disappeared. However, cortical atrophy and an enlargement of the ventricles were still present. In an asymptomatic girl, the cranial CT scans remained normal after chelating therapy. The cranial CT scan is considered to be of great use both in diagnosis and observation of the clinical course in Wilson disease, and it is also of prognostic value.

Cytochrome c oxidase deficiency in Menkes kinky hair disease.

In a 4-year-old male with Menkes kinky hair disease (MKHD) treated with copper supplement therapy, reduced cytochrome a + a3 contents in liver was demonstrated to be 0.029 against 0.128 nmol/mg protein in the control. Cytochrome c oxidase activities in brain, liver, skeletal muscle, and heart were 47, 22, 54 and 59% of the control, respectively. The copper contents in brain and liver were decreased. In spite of increased serum levels of copper and ceruloplasmin, the decreased cytochrome c oxidase activities in various organs were not corrected by copper supplement therapy. A search for a therapeutic method which can normalize copper enzymes in brain and liver, would seem to be a prerequisite for the treatment of MKHD.

Hyperargininemia: clinical course and treatment with sodium benzoate and phenylacetic acid.

In a patient with hyperargininemia, oral administration of sodium benzoate or phenylacetic acid together with an essential amino acid mixture was used to prevent hyperammonemia and to decrease plasma and CSF concentrations of arginine. Sodium benzoate reduced the plasma ammonia levels, which was confirmed by the increase of urinary excretion of hippuric acid. Phenylacetic acid also controlled hyperammonemia, and EEG findings also improved. By these treatments, plasma and CSF concentrations of arginine showed a slight decrease, but were far above the normal range. There was no clinical improvement, and spasticity of the lower and upper extremities was progressive with mental deterioration.

Seizure prognosis and EEG evolution in complex partial seizures of childhood onset.

We studied the clinical course and seizure prognosis of 126 children with complex partial seizures regularly followed up for more than 4 years in our clinic. Clinical and EEG features of 63 seizure-free patients were compared with those of 63 patients with persistent seizures. The features contributing to poor prognosis were 1) mental retardation, 2) a history of status epilepticus and 3) abnormal basic rhythm in EEG. CT abnormality, a history of febrile convulsions (FC), the clustering of seizures and association with other types of seizures did not influence prognosis. We divided the patients into four groups according to the evolutionary pattern of seizure discharges: Group A, 55 (43.7%) patients with spike focus always fixed in the same region; Group B, 20 (15.9%) patients with wandering foci; Group C, 10 (7.4%) patients with multifocal spikes; and group D, 41 (32.5%) patients with no focal discharges. There was no difference in seizure prognosis among these four groups, but the patients with a focus in the anterior temporal region in Group A evidenced the worst prognosis.

Guillain-Barré syndrome presenting with signs of ophthalmoplegia.

Two cases of Guillain-Barré syndrome (GBS) presenting with signs of ophthalmoplegia were reported. One showed unilateral internal ophthalmoplegia without external ophthalmoplegia, and the other displayed partial external ophthalmoplegia with sluggish pupillary response to light. It is important to keep in mind the possibility of GBS with acute onset of internal and/or external ophthalmoplegias.

Moyamoya disease presenting with chorea.

Three children with moyamoya disease are reported whose initial and predominant manifestations were choreic movements. Two of the patients presented with unsteady gait and the other with clumsiness. Choreic movements were recurrent and were often triggered by excitement, emotional tension, or crying. They occurred unilaterally or bilaterally and often alternated between the right and left. Moyamoya disease must be considered in the differential diagnosis of acquired chorea in children.

Development of the beta activities in children: an EEG-topographical analysis.

We investigated the characteristics of beta activities during drowsiness and very light sleep in 61 children using topographical analysis. The average power of beta activities was posterior-dominant in infancy, and it became frontal-dominant as the age increased. These topographical changes occurred earlier in the low frequency bands than the high frequency bands. The total power of the high frequency bands was larger in infants, and with increasing age that of the low frequency bands predominated. The developmental changes of the beta activities considered to be induced by drugs were similar to those of children without drugs.

Antiepileptic drugs--calcium current interaction in cultured human neuroblastoma cells.

The voltage-dependent calcium channel current (ICa) in the neuroblastoma cell line of human origin (NB-I) was studied by the whole-cell clamp recording. Three types of ICa were identified in NB-I cells. Our electrophysiological and pharmacological findings have suggested that these three types of ICa are consistent with the T-, N- and L-type ICa, respectively. Phenytoin (PHT) inhibited T-type ICa by 13.0% at a concentration of 5 microM, and L-type ICa by 6.3% at a concentration of 100 microM. At a concentration of 100 microM, carbamazepine (CBZ) inhibited T- and L-type ICa by 6.0% and 5.9%, respectively. At a concentration of 50 microM, sodium valproate (VPA) blocked T- and L-type ICa by 6.1% and 47.5%, respectively. At a concentration of 50 microM, zomisamide (ZNS) inhibited T- and L-type ICa by 38.3% and 41.9%, respectively. Na+ channel blockade has been reported to be responsible for the clinical efficacy of PHT or CBZ. Inhibition of T-type ICa by PHT may enhance the efficacy of its anticonvulsant action. CBZ had little effect on ICa. The anticonvulsant activity may be related to the blockade of T-type ICa in the case of VPA and ZNS.