A case of neonate effectively treated with everolimus for giant hepatic hemangioma complicated with congenital duodenal atresia and Kasabach-Merritt syndrome.

BACKGROUND: Disseminated intravascular coagulation (DIC) with Kasabach-Merrit syndrome from a large hepatic hemangioma is life-threatening. We report a case of giant hepatic hemangioma of the newborn with KMS. RESULTS: The patient was born at 37 gestational weeks and 2 days via cesarean section; weight at birth was 2952 g. Congenital duodenal atresia was noted during the fetal period. DIC developed after delivery and a giant liver hemangioma was diagnosed via abdominal CT. The cause of DIC was Kasabach-Merritt syndrome owing to a giant hepatic hemangioma. First, combination therapy of 2 mg/kg/day of prednisolone and 0.2 mg/kg/day of propranolol was initiated form enterostomy. However, the size of the hepatic hemangioma did not alter, as observed via image evaluation. Therefore, 0.3 mg/kg/day of everolimus was administered frorm enterostomy. Subsequently, the size of the hepatic hemangioma was assessed via image evaluation. Although it did not alter, blood flow to the hepatic hemangioma decreased and thrombocytopenia was also suppressed. We performed hepatic lateral segmentectomy, radical operation for duodenal atresia. The pathological diagnosis of the removed tumor was infantile hemangioma. CONCLUSION: We report everolimus may be useful when PSL and propranolol are ineffective.

Association of a basic 25K protein with membrane coating granules of human epidermis.

Keratinocytes of the upper granular layers contain unique round-to-oval granules, 100-500 nm in diameter, in their peripheral cytoplasm. These granules (known as membrane coating granules [MCG], or lamellar granules) fuse with the apical cell surface of uppermost granular cells and discharge their contents into the intercellular space, where they are believed to play a role in establishing the permeability barrier of the epidermis and possibly in regulating the orderly desquamation of terminally differentiated keratinocytes. Using two monoclonal antibodies originally prepared against hair follicle antigens, we have identified a 25K epidermal protein in association with both MCG-like granules in the peripheral cytoplasm of granular cells as well as MCG-derived intercellular material. This protein is relatively basic (pI greater than 8), largely aqueous soluble, methionine deficient, and is relatively abundant in epidermis (comprising up to approximately 0.1% of soluble proteins). Its distribution is restricted to the granular layer of keratinized (cornified) stratified squamous epithelia. The identification of this protein component opens new avenues for studying the molecular mechanisms underlying the establishment of permeability barrier and/or regulation of desquamation.

Uroplakin I: a 27-kD protein associated with the asymmetric unit membrane of mammalian urothelium.

The luminal surface of mammalian urothelium is covered with numerous plaques (also known as the asymmetric unit membrane or AUM) composed of semi-crystalline, hexagonal arrays of 12-nm protein particles. Despite the presumed importance of these plaques in stabilizing the urothelial surface during bladder distention, relatively little is known about their protein composition. Using a mouse mAb, AE31, we have identified a 27-kD protein that is urothelium-specific and is differentially expressed in superficial umbrella cells. This protein (pI approximately 5.8) partitions into the detergent phase during Triton X-114 phase separation. Pulse-chase experiments using cultured bovine urothelial cells showed that this protein is synthesized as a 32-kD precursor that is processed through a 30-kD intermediate, to the mature 27-kD form. In cytoplasmic vesicles containing immature AUM, the AE31 epitope is detected in patches on the cytoplasmic side, but in mature, apical AUM it is detected exclusively on the luminal side. This suggests an unusual translocation of the AE31 epitope during AUM maturation; more data are required, however, to substantiate this interpretation. Immunoaffinity purification of the 27-kD protein results in the copurification in approximately molar ratio of a 15-kD protein, as well as a small and variable amount of a 47-kD protein. Immunoblotting data indicate that these three proteins are immunologically distinguishable. This copurified 15-kD protein is relative basic (pI approximately 8.0). Like the 27-kD protein, it is urothelium-specific and is present mainly in the umbrella cells. Together, our data indicate that a 27-kD protein is urothelial plaque-associated (uroplakin I). Based on complex formation data, we provisionally name the 15-kD protein uroplakin II; additional data will be required to determine whether this and the 47-kD protein are integral parts of AUM. The identification of these AUM-associated and -related proteins, plus the availability of a culture system capable of synthesizing and processing some of these molecules, offer new opportunities for studying the detailed structure, assembly, and function of asymmetrical unit membrane.

Accuracy and reliability of continuous blood glucose monitor in post-surgical patients.

BACKGROUND: The STG-22 is the only continuous blood glucose monitoring system currently available. The aim of this study is to determine the accuracy and reliability of the STG-22 for continuously monitoring blood glucose level in post-surgical patients. METHODS: Fifty patients scheduled for routine surgery were studied in surgical intensive care unit (ICU) of a university hospital. After admission to the ICU, the STG-22 was connected to the patients. An attending physician obtained blood samples from a radial arterial catheter. Blood glucose level was measured using the ABL800FLEX immediately after blood collection at 0, 4, 8, and 16 h post-admission to the ICU (total of 200 blood glucose values). RESULTS: The correlation coefficient (R2) was 0.96. In the Clarke error grid, 100% of the paired measurements were in the clinically acceptable zone A and B. The Bland and Altman analysis showed that bias+/-limits of agreement (percent error) were 0.04(0.7)+/-0.35(6.3) mmol (mg/dl) (7%), -0.11(-2)+/-1.22(22) (15%) and -0.33(-6)+/-1.28(23) (10%) in hypoglycemia (<70(3.89) mmol (mg/dl), normoglycemia (3.89(70)-10(180) mmol (mg/dl), and hyperglycemia (>10(180) mmol (mg/dl), respectively. CONCLUSIONS: The STG-22 can be used for measuring blood glucose level continuously and measurement results are consistent with intermittent measurement (percentage error within 15%). Therefore, the STG-22 is a useful device for monitoring in blood glucose level in the ICU for 16 h.

Bleeding time prolonged by daily low-dose aspirin is shortened by one medium dose aspirin.

BACKGROUND: In planning surgery, a low-dose aspirin regimen for prevention of thrombotic events is often discontinued in order to avoid the risk of excessive bleeding during surgery. However, this procedure increases the risk from adverse thrombotic events. We propose a different method, which may normalize the prolonged bleeding time caused by low-dose aspirin. We verified the effectiveness of this method in healthy volunteers. METHODS: Volunteers with bleeding time prolonged by taking 81 mg of aspirin a day for a period of 1 week were randomly divided into two groups. The test group of 18 volunteers received a dose of 660 mg of aspirin, while the control group of 16 received placebo. Bleeding time and maximum platelet activity were then evaluated. RESULTS: Before 660 mg of aspirin or placebo, bleeding time was prolonged: in the aspirin group from 3.1 +/- 0.7 to 6.1 +/- 1.4 min (n=18), and in the placebo group from 2.9 +/- 0.9 to 6.1 +/- 1.5 min (n=16). This prolongation was significant in both groups at the P<0.01 level. In the test group, bleeding time was shortened to 4.5 +/- 1.3 min (P<0.01), which is in the normal range, while it remained prolonged in the control group (6.0 +/- 1.2 min). Platelet activity, on the other hand, was suppressed in both groups. CONCLUSION: We conclude that 660 mg of aspirin effectively shortens the bleeding time prolonged by daily low-dose (81 mg) aspirin.

Distribution of immunoreactive malondialdehyde-modified low-density lipoprotein in human serum.

We developed a sensitive enzyme-linked immunosorbent assay (ELISA) for detection of malondialdehyde-modified low-density lipoprotein (MDA-LDL) in human serum. A monoclonal antibody against MDA-LDL (ML25) used in our method recognized not only MDA-LDL but also other MDA-modified proteins. However, MDA-LDL was able to be detected specifically by using a combination of ML25 and an antibody specific for apolipoprotein B (apo B) (AB16), which was conjugated with beta-galactosidase. Using this method, measurable amounts of MDA-LDL were detected in the sera of 40 healthy individuals. MDA-LDL was observed to be mainly distributed in the human LDL fraction separated by density gradient ultracentrifugation, while in each lipoprotein subfraction the largest amount of MDA-LDL per protein was found at a subfraction between LDL and HDL. The particle size of LDL in this fraction was smaller than that of LDL in the main LDL fraction, as assessed by electrophoresis. In addition, LDL oxidized by Cu2+ was also detectable with this method. We conclude that our method is sensitive and specific for MDA-LDL and might be useful for investigating MDA-LDL in the human circulation.

Mock-up experiment at Birmingham University for BNCT project of Osaka University--Neutron flux measurement with gold foil.

Mock-up experiment for development of accelerator based neutron source for Osaka University BNCT project was carried out at Birmingham University, UK. In this paper, spatial distribution of neutron flux intensity was evaluated by foil activation method. Validity of the design code system was confirmed by comparing measured gold foil activities with calculations. As a result, it was found that the epi-thermal neutron beam was well collimated by our neutron moderator assembly. Also, the design accuracy was evaluated to have less than 20% error.

A cluster of lysinuric protein intolerance (LPI) patients in a northern part of Iwate, Japan due to a founder effect. The Mass Screening Group.

Lysinuric protein intolerance is an autosomal recessive disease characterized by defective transport of the dibasic aminoacids. Mutational analysis of LPI patients in the northern part of Japan revealed that six were homozygous for the R410X mutation and two others were compound heterozygotes of R410X and other unknown mutations. In the population epidemiology study in a local cluster in the northern part of Iwate, ten heterozygotes were found in 1190 newborn babies leading to an estimated LPI incidence of 1/57,000. Polymorphism analysis revealed two major alleles, A and B, in intron 8. While the population frequency of allele A was 0.9 and that of allele B was 0.1 in the northern part of Japan the R410X mutations were exclusively on allele B in 31 chromosomes suggesting a founder effect. Genetic analysis in patients revealed strong linkage disequilibrium with D14S283 and TCRA indicating that the R410X mutation occurred before at least 130 generations ago (about 2600 years). The R410X mutation was shown to be useful as a molecular marker for screening LPI patients in the northern part of Japan.

In vitro damage of basal lamina-associated anionic sites by hydroxyl radical.

We examined the effect of chemically generated hydroxyl radical on basal lamina-associated anionic sites. Cytochemical studies showed that hydroxyl radical produced a loss of cationic tracer-positive, anionic particles, and this effect was inhibited by a specific scavenger, thiourea. These data might suggest that anionic sites were degraded by hydroxyl radical which was derived, for example, from the activated leukocytes in close contact with the dermal-epidermal junction during acute inflammation resulting in the disturbance of the charge-selective permeability barrier.

Ultrastructural demonstration of anionic sites in basement membrane zone by cationic probes.

Anionic sites were demonstrated ultrastructurally in both the dermal and epidermal edges of the lamina densa using strongly cationized polyethyleneimine as a tracer. Enzyme digestion with heparitinase and pronase removed the anionic binding sites, indicating that the sites consisted largely of heparan sulfate proteoglycan.

Expression of anionic sites at the dermoepibolic junction.

The emergence of anionic sites during basement membrane zone formation was studied using migrating epidermis in organ culture as a model system. Ultrastructural investigations using a strongly cationized probe revealed that the heparitinase-sensitive, anionic sites were formed synchronously with the newly built basal lamina after 7 days in culture.

Interaction of trichohyalin with intermediate filaments: three immunologically defined stages of trichohyalin maturation.

"Trichohyalin" is a 220-kD protein found in trichohyalin granules that are present as major differentiation products in the medulla and inner root sheath cells of human hair follicles. It was unclear whether this protein served as an intermediate filament precursor in the inner root sheath or as an intermediate-filament-associated (matrix) protein. We have produced a panel of monoclonal antibodies (AE15-17) to this protein and used them to trace its fate during inner root sheath differentiation. These studies have allowed us to define three immunologically distinct forms of this trichohyalin protein. They are 1) the AE15-positive form, which is found throughout all trichohyalin granules; 2) the AE16-positive form, which is localized as discrete punctae on the surface of trichohyalin granules; and 3) the AE17-positive, intermediate-filament-bound form, which associates with the inner root sheath filaments with a regular, 400-nm periodicity. From these results we suggest that the 220-kD trichohyalin protein is an intermediate-filament-associated protein that may play a role in the lateral aggregation, precise alignment, and stabilization of inner root sheath filament bundles.

Production of blister in normal human skin in vitro by blister fluids from epidermolysis bullosa.

Pathogenic mechanisms involved in the blister formation of epidermolysis bullosa (EB) are not clearly understood at present. In this paper, we attempted to produce experimental blistering in vitro similar to the histologic picture of EB simplex (EBS) and recessive dystrophic EB (RDEB). It was demonstrated by light and electron microscopy that the medium containing fresh blister fluids from the patients with EBS or RDEB could produce similar histologic features in normal human skin (in vitro) to those of the skin lesions of patients (in vivo). This observation may open new avenues of approach to studying these diseases.

Detection of deiminated proteins in rat skin: probing with a monospecific antibody after modification of citrulline residues.

We performed a systematic study on deiminated proteins present in rat epidermis. Proteins extracted from various epidermal samples were resolved by either one- or two-dimensional gel electrophoresis and Western blotted to nitrocellulose membranes. Deiminated proteins were detected by modification of citrulline residues followed by probing with an anti-modified citrulline monospecific antibody. The cornified layer of adult plantar skin gave multiple series of isoelectric variants, most of which were found to be differentially deiminated type II keratins (60 kDa, and 67 kDa or above). The whole epidermis of 5-day-old rat back skin showed isoelectric variants of 60-kDa keratin as major deiminated components, and deiminated 55-kDa keratin and deiminated filaggrin as minor spots. In addition, we found highly deiminated proteins (200-220 kDa) thought to be derived from trichohyalin. The immunoreactivity of deiminated proteins was mainly localized in the granular and cornified layers of epidermis. Co-localization of deiminated filaggrin and keratins in the granular layer suggests the possible role of protein deimination during the terminal stage of epidermal differentiation.

Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.

Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.

Axonal trajectory and terminal distribution of inspiratory neurons of the dorsal respiratory group in the cat's medulla.

In Nembutal-anesthetized and artificially ventilated cats, we studied the morphological properties of the inspiratory neurons of the dorsal respiratory group (DRG) with HRP intracellular staining. A total of 37 neurons were stained and their axonal trajectories and terminal distribution in the medulla were analyzed. The somata were located predominantly in the ventrolateral region of the nucleus of the solitary tract and were distributed between 2,300 mum rostral and 700 mum caudal to the obex. Most (26/33) of the neurons tested were antidromically activated by the stimulation of the contralateral (n = 24) or ipsilateral (n = 2) cervical cord. To examine the existence of collateral branches in the brainstem, we traced axonal trajectories in 28 neurons. In most cases, the stem axons issuing from the cells of origin coursed ventrally and then turned medially to cross the midline without giving off any axon collaterals. However, six neurons had axonal collaterals in the brain stem ipsilateral to the somata. At least four types of collateralization were observed. The stem axon of the first type bifurcated at the area ipsilateral and ventral to the cell body. One branch crossed the midline to project to the spinal cord, and the other, thinner branch descended caudally in the ipsilateral medullary reticular formation without distributing any terminals. The axon of the second type projected to the contralateral spinal cord and distributed collateral branches with terminal boutons in the ipsilateral ventral respiratory group (VRG). The third type projected to the contralateral spinal cord and distributed terminal boutons in the medial part of the nucleus of the solitary tract (NTS) and its vicinity. The fourth type distributed numerous branches with terminal boutons in and around the ventral part of the NTS and the VRG area. This study indicates that some inspiratory neurons of the DRG influence not only spinal respiratory neurons but also medullary respiratory neurons in the vicinity of the DRG and the VRG.

Medullary projection of nonaugmenting inspiratory neurons of the ventrolateral medulla in the cat.

In Nembutal-anesthetized, immobilized, and artificially ventilated cats, we studied the morphological characteristics of inspiratory neurons with nonaugmenting firing patterns. HRP was injected intracellularly into a total of 22 neurons of the Bötzinger complex (BOT) and the ventral respiratory group (VRG). In 20 cases somata with their axonal trajectories were stained, and in two cases only axons were stained. None of the neurons stained could be antidromically activated by stimulation of the cervical cord. The somata of 20 neurons were located in the vicinity of the nucleus ambiguus or the retrofacial nucleus (RFN) between 600 microns and 2,800 microns caudal to the rostral end of the RFN. Their axons could be traced for a distance of several millimeters on the side of the somata, and showed various projection patterns. According to these projection patterns, the 20 neurons were tentatively classified into four groups: A (8/20), B (4/20), C (6/20), and motoneurons (2/20). Group A neurons gave off extensive axon collaterals that arborized and distributed boutons predominantly in the BOT and the VRG areas. Group B neurons had less extensive axon collaterals with various projection patterns, projecting rarely to the BOT or the VRG area. Group C neurons sent their stem axons, without issuing any axonal collaterals, to the contralateral side in five cases and to the ipsilateral pons in one case. The two motoneurons had axons leaving the brainstem without any intramedullary collaterals. Thus, the nonaugmenting inspiratory neurons showed morphological variations, which may play different roles in neural control of respiration.

Morphology of augmenting inspiratory neurons of the ventral respiratory group in the cat.

The present study examined, in Nembutal-anesthetized and artificially ventilated cats, the morphologic properties of the inspiratory neurons of the ventral respiratory group (VRG). Horseradish peroxidase (HRP) was injected into 21 augmenting inspiratory or late inspiratory neurons with peak firing rates in the late inspiratory phase. The majority of the stained neurons were antidromically activated by stimulation of the cervical cord. Thirteen somata, located within or around the nucleus ambiguus (AMB), between 100 microns caudally and 2,000 microns rostrally to the obex, were stained. In ten cases, the stem axons issuing from the cells of origin coursed medially to cross the midline without giving off any axonal collaterals. Three neurons gave rise to axonal collaterals on the ipsilateral side, distributing boutons in the medullary reticular formation, in the vicinity of the AMB, hypoglossal nucleus, solitary tract, and dorsal motor nucleus of the vagus. In eight neurons, only the axons were labeled; in four of these, which were antidromically activated from the spinal cord, the stem axons crossed the midline 2,000-3,000 microns rostral to the obex and descended in the reticular formation around the AMB down to the cervical cord. They issued several axonal collaterals, distributing terminal boutons at the level of the caudal end of the retrofacial nucleus and about 1,000 microns rostral and caudal from the obex. Terminals were found mainly in and around the AMB, and a few were found in the vicinity of the dorsal motor nucleus of the vagus. The remaining four nonactivated axons distributed their terminal boutons widely in the reticular formation around the AMB. Thus, the augmenting inspiratory neurons of the VRG were shown to project not only to the spinal cord, but also to the VRG, hypoglossal nucleus, and dorsal motor nucleus of the vagus.