Clinical pictures of 75 patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).

We clarified the clinical features of NICCD (neonatal intrahepatic cholestasis caused by citrin deficiency) by retrospective review of symptoms, management and long-term outcome of 75 patients. The data were generated from questionnaires to paediatricians in charge of the patients. Thirty of the patients were referred to hospitals before 1 month of age because of positive results in newborn screening (hypergalactosaemia, hypermethioninaemia, and hyperphenylalaninaemia). The other 45, the screen-negative patients, were referred to hospitals with suspected neonatal hepatitis or biliary atresia because of jaundice or discoloured stool. Most of the screen-negative patients presented before 4 months of age, and 11 had failure to thrive. Laboratory data showed elevated serum bile acid concentrations, hypoproteinaemia, low levels of vitamin K-dependent coagulation factors and hypergalactosaemia. Hypoglycaemia was detected in 18 patients. Serum amino acid analyses showed significant elevation of citrulline and methionine concentrations. Most of the patients were given a lactose-free and/or medium-chain triglyceride-enriched formula and fat-soluble vitamins. Symptoms resolved in all but two of the patients by 12 months of age. The two patients with unresolved symptoms suffered from progressive liver failure and underwent liver transplantation before their first birthday. Another patient developed citrullinaemia type II (CTLN2) at age 16 years. It is important to recognize that NICCD is not always a benign condition.

Rapid disappearance of the medial epithelial seam during palatal fusion occurs by multifocal breakdown that is preceded by expression of alpha smooth muscle actin in the epithelium.

Breakdown of the medial epithelial seam (MES) is essential to allow bridging of the mesenchyme during palatal fusion. Evidence exists for three mechanisms for this breakdown that are incompatible at the level of individual cells in the seam. To determine if breakdown of the seam was regionally restricted, 3-dimensional reconstructions were generated using volume rendering software from 1 micron serial sections in the sagittal plane of rat palates fixed during the process of fusion. The earliest break detected in electron micrographs was cell separation and in reconstructions was a discrete defect, with a rounded outline, nearer to the nasal than to the oral margin of the seam. Further breakdown produced a pattern of rounded defects along the nasal margin of the seam resulting in interconnected columns of cells preferentially attached to the oral epithelium. Computer generated slicing of reconstructed seams showed that groups of cells evident in cross-sections as islands at this stage of breakdown of the MES could be artifacts. Unequivocal islands of epithelial cells formed later in fusion had a rounded outline, an incomplete basal lamina and a halo of cells containing phagocytosed apoptotic debris. The pattern of breakdown indicated that the MES breaks down under tension. Laser confocal microscopy of sections and whole-mounts of palates demonstrated alpha-smooth muscle actin preferentially localized in the epithelial cells of the palatal shelves immediately before and during formation of the seam. Expression in epithelial cells of the isoform of actin normally restricted to smooth muscle cells engaged in tonic contraction supported an interpretation that the epithelial cells of the seam may be capable of generating tension during the palatal fusion event.

Midline fusion in the formation of the secondary palate anticipated by upregulation of keratin K5/6 and localized expression of vimentin mRNA in medial edge epithelium.

Secondary palatal fusion is dependent on targeted removal of the epithelium between the palatal shelves. Aseptically delivered rat embryos 15 through 18 days post coitum (dpc) were probed with DIG-labeled antisense and sense ssDNA probes for spliced exon sequences flanking intron E of cytokeratins K5/6 and spliced exon sequences flanking intron F of vimentin. Cytokeratin K5/6 expression was upregulated in the medial edge epithelium (MEE) prior to rotation of the palatal shelves and in the vomerine epithelium in the region of fusion with the palate. K5/6 expression continued in the medial epithelial seam (MES) and in epithelial islands during breakdown of the MES. Vimentin expression was not detected in the MEE prior to rotation but was specifically upregulated in the MEE following rotation and prior to midline contact and continued in the MES and in epithelial cells identifiable during the breakdown of the MES. Initiation of vimentin upregulation in the MEE prior to contact of the palatal shelves was tested by serum-free organ culture of palates from embryos at 15.5 dpc with the shelves separated by a biocompatible membrane. Vimentin upregulation occurred in the epithelium specifically in the region of anticipated contact. These results are interpreted as indicating that i) cytokeratin K5/6 expression may play a critical role in the integration of the epithelial layers of the MES to ensure subsequent merging of the mesenchyme and ii) epithelial cells in the MEE are specifically 'primed' to upregulate expression of mesenchymal genes prior to integration into and breakdown of the MES.

Serum vitamin E levels in children with corrected biliary atresia.

In 37 children with long-standing cholestasis who had undergone a Kasai's procedure (double Roux-en-Y hepatic portoenterostomy), serum vitamin E levels were determined. In addition, serum bile acid levels were simultaneously tested as a marker of cholestasis. Eighteen of 37 children had vitamin E levels of less than 0.50 mg/100 ml, and two showed neurological abnormalities including hypoactive deep tendon reflexes and ataxia. Serum vitamin E levels were inversely correlated with serum bile acid levels (p less than 0.01). Older patients have mild cholestasis and high serum vitamin E levels in comparison with younger ones. Improvement in bile excretion into the intestinal tract with age seemed to be responsible for an increase of serum vitamin E levels. Oral supplements of alpha-tocopherol in doses of 5 to 10 mg/kg/day were needed to maintain the normal serum vitamin E levels in postoperative infants.

A case of fatal infectious mononucleosis presenting with fulminant hepatic failure associated with an extensive CD8-positive lymphocyte infiltration in the liver.

We describe a fatal case of infectious mononucleosis presenting with fulminant hepatic failure associated with extensive CD8-positive lymphocyte infiltration and diffuse karyorrhexis in the liver. Immunohistochemical analysis of mononuclear cells showed that Leu-2a (CD8)-positive lymphocytes were heavily distributed in the portal areas and the sinusoidal spaces, but Leu-3a (CD4)-, Leu-14 (CD22)-, or My 4 (CD14)-positive cells were undetectable in sections of the liver. Southern blot hybridization studies disclosed the presence of Epstein-Barr virus DNA fragments in the liver tissue. The unusual pathologic and immunologic responses observed in this case could not simply be explained by severe Epstein-Barr virus infection. Some superimposed factors should be considered.

Heterogeneity of liver disorder in type B Niemann-Pick disease.

Patients with type B Niemann-Pick disease (NPD) are known to be complicated with varying degrees of prognosis-determining liver dysfunction. To see heterogeneity of the dysfunction histologically, we performed liver biopsies on three NPD patients from three different families, who were diagnosed by enzyme assay of acid sphingomyelinase (ASM) and analysis of the ASM gene. In a severe case, of a female patient in her childhood, the liver showed definite fibrosis despite her age. In contrast, in a very mild case, of an adult male patient, the liver showed little fibrosis, though the ballooning of hepatocytes and infiltration of foamy histiocytes were observed in the tissue. Three homo-allelic mutations (S436R, A599T, and S231P) were identified in the patients. Thus, various hepatic phenotypes in type B NPD were shown to be caused by the heterogeneity of liver lesions originating from different ASM gene mutations.

Occurrence of both (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acids in urine from an infant with Zellweger's syndrome.

Urine from a patient with Zellweger's syndrome was examined for bile acids after fractionation into three groups according to mode of conjugation. 3 alpha,7 alpha,12 alpha-Trihydroxy-5 beta-cholestanoic acid was the predominant bile acid of the unconjugated and glycine-conjugated bile acid fractions. Smaller amounts of cholic acid and 1 beta-, 6 alpha-, 24-, and 26-hydroxylated derivatives of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid were found in both fractions in similar proportions. The bile acid spectrum of the taurine-conjugated bile acid fraction was different from those of the other two fractions in the occurrence of two new compounds as the major constituents. These compounds were tentatively identified as two epimers at C-23 of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestano-26,23-lactone, which were probably artifacts formed from the corresponding tetrahydroxycholestanoic acids during the procedures for extraction after hydrolysis. High-performance liquid chromatographic analysis revealed that 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid excreted into the urine as the unconjugated form consisted of a mixture of (25R)- and (25S)-isomers in the ratio of about 7:3.

Olfactory input to the lateral hypothalamus of the old world monkey.

Responses of lateral hypothalamic neurons to 8 odors were studied in chronic unanesthetized old world monkeys (Macaca irus). Many neurons (54.5%) responded to a single odor only, and the number of neurons responding to 2, 3 and 4 odors decreased successively. No neuron responded to as many as 5 odors. Thus, the presence of olfactory input and a highly discriminative ability for odors were found in the lateral hypothalamic area (LHA). Neuronal responses to the same odors were also studied in the septum (Spt). In anesthetized old world monkeys, evoked potentials were recorded in the LHA and in areas of the Spt and the nucleus accumbens (Acc) during stimulation of the olfactory bulb (OB). When the Spt (and probably the Acc with it) was subsequently destroyed, OB-evoked potentials in the LHA disappeared. Next, by injecting horseradish peroxidase (HRP) into the LHA, an olfactory pathway to the LHA was examined. Labeled neurons were found mainly in the Spt and the Acc, and only partly in other areas. However, labeled neurons were scarcely found in the prepyriform (PPF)-entorhinal (ER) area or in the olfactory tubercle (OT). The present study thus shows that an olfactory pathway to the LHA passes through the Spt and probably also the Acc, but not through the PPF-ER areas nor through the OT in the old world monkey.

Projections of extraocular muscle primary afferent neurons to the trigeminal sensory complex in the cat as studied with the transganglionic transport of horseradish peroxidase.

The central projections of extraocular muscle primary afferent neurons were examined in the cat by means of transganglionic axonal transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Injections of the extraocular muscle with WGA-HRP resulted in transganglionic terminal labeling within the ipsilateral trigeminal sensory complex. Although the density of trigeminal projections varied among cases, labeled axons and terminals were heavily and consistently found within the rostroventral portion of the pars oralis of the spinal trigeminal nucleus. The caudal part of the trigeminal principal sensory nucleus occasionally contained moderate labeling but very few deposits of HRP reaction product were noted in the pars interporalis and pars caudalis of the spinal trigeminal nucleus.

Urinary bile alcohol profiles in healthy and cholestatic children.

BACKGROUND: Bile alcohols are normal constituents of urine. METHODS: To better understand bile alcohol profile in childhood, urinary specimens from 41 healthy children and 10 children with cholestasis, and 3 healthy adults, were analyzed by GLC and GC-MS. RESULTS: Five bile alcohols, 27-nor-5beta-cholestane-3alpha,7alpha,12alpha,24S,25R-pentol, 5beta-cholestane-3alpha,7alpha,12alpha,24S, 25-pentol, 5beta-cholestane-3alpha,7alpha,12alpha,24S,26-pentol, 5beta-cholestane-3alpha,7alpha, 12alpha,25,26-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,26,27-pentol were identified in all specimens. C(26)-Pentol was the most abundant constituent, constituting 29.5 to 65% of bile alcohols. Among healthy children (n=41), no significant relationship was seen between proportions of the C(26)-pentol and age, but older children (n=15, 6 to 14 years) showed a significantly greater mean percentage of the C(26)-pentol than young children (n=26, 0 to 5 years; 58.1+/-4.23% vs. 46.0+/-9.24%, p<0.001). In children with cholestatic liver diseases, the percentage of C(26)-pentol in urinary bile alcohols was significantly lower than age-matched controls. CONCLUSIONS: There is an increased composition of C(26)-pentol in older children and relatively decreased composition of C(26)-pentol in children with cholestatic liver diseases.

An undescribed subset of neonatal intrahepatic cholestasis associated with multiple hyperaminoacidemia.

Five patients of cholestatic jaundice and multiple hyperaminoacidemias were uncovered during neonatal mass screening for homocystinuria. All five patients had increased plasma levels of methionine, citrulline, tyrosine, threonine, phenylalanine, lysine and arginine. Compared with those of age-matched cholestatic disease controls, idiopathic neonatal hepatitis (n=9) and biliary atresia (n=14), plasma levels of three amino acids, citrulline, methionine, and threonine, were significantly greater, respectively (P<0.01). Liver biopsies examined in four patients uniformly showed diffuse hepatic fatty liver with micro- and macrovesicular droplets without giant cell transformation. Administration of fat-soluble vitamins and formula milk containing middle-chain triglyceride resulted in normalization of amino acid profiles by 6 weeks after the treatment. All liver function tests normalized by 17 months of age.

Nerve regeneration in cornea after penetrating keratoplasty in rabbit, with special reference to relationship between regenerating nerves and basal laminae.

The pattern of nerve regeneration in the grafted rabbit cornea was investigated by electron microscopy. Grafted corneas were excised 2, 7, 14 and 28 days after grafting, and processed for observation by conventional electron microscopy. In the normal, unoperated cornea Schwann cell basal laminae are, unlike those of ordinary peripheral nerves, discontinuous and fragmentary on the fibers coursing through the corneal stroma. In the early stage of regeneration, while numbers of regenerating axons extended through the Schwann cell columns of regenerating axons extended through the Schwann cell columns in the grafts, many other regenerating axons elongated as single fibers through the corneal stroma outside the Schwann cell columns. These single naked axons were later enveloped by Schwann cell cytoplasm, contributing to the overall dense irregular pattern of regenerated nerves in the grafted cornea. It was thought that the regenerating axons can extend throughout the stroma without the guidance of basal lamina tubes, making the corneal stroma a favorable environment for nerve regeneration.

Long-term follow-up of excimer laser phototherapeutic keratectomy.

PURPOSE: To evaluate long-term follow-up results of excimer laser phototherapeutic keratectomy (PTK) in a Japanese population. METHODS: Twenty-six patients (31 eyes) with corneal opacity were treated with excimer laser PTK. Preoperative diagnoses included 16 eyes with band keratopathy, 10 with granular dystrophy, and 5 with corneal scar. Mean postoperative follow-up was 27 months. RESULTS: Corneal opacity was reduced in all patients. At postoperative month 12, best spectacle-corrected visual acuity (BSCVA) improved from the preoperative level in 22 eyes of 28 eyes, did not change in 3 eyes, and declined in 3 eyes. BSCVA at month 24 was better than the preoperative acuity in 17 eyes of 23 eyes, similar in 1 eye, and worse in 5 eyes. Eyes with granular dystrophy showed significantly better BSCVA improvement than those with band keratopathy. A hyperopic shift of +1.0 diopter or more occurred in 14 eyes of 28 eyes at month 12 and in 12 eyes of 23 eyes at month 24. No serious adverse effects were encountered during the 3-year follow-up period. CONCLUSIONS: Excimer laser PTK is a safe and effective procedure for the treatment of Japanese patients with superficial corneal opacity.

Clinical application of indocyanine green angiography to choroidal neovascularization.

In order to evaluate the clinical usefulness of indocyanine green video-angiography (IA), the angiographic features of choroidal neovascular membranes (CNM) were investigated in 27 eyes with choroidal neovascular diseases by means of standard fluorescein angiography (FA) and IA. FA showed the existence of CNM in 21 eyes and IA demonstrated evidence of CNM in 19 eyes, as "fan, comb or spotty hyperfluorescence" in the early stage and "leakage" in the late stage. In 6 out of 19 eyes the existence of CNM was shown by IA, while FA failed to identify the precise location and size of CNM due to the masking effect of overlying turbid fluid, massive hemorrhage or a large amount of serous fluid. The results imply that IA has an advantage over FA in cases where FA shows only the sign of occult choroidal neovascularization, and that IA can be applied to neovascular maculopathy as a routine examination.

ERG c-wave and pigment granule distribution in isolated chick retina: effects of osmotic pressure variation in vitreal perfusing solution.

The relation between osmotic gradient variation and change in the ERG c-wave was investigated. The vitreous side of the isolated chick retina was perfused with various solutions having osmotic pressures (pi v) ranging between 310 and 600 mOsm, while the choroidal side was maintained at 480 mOsm (pi c). When the osmotic difference (delta pi), (ie, pi v-pi c), was larger than -10 mOsm, normal c-waves with slow rise-times (cs-wave) were obtained; however, when the delta pi was smaller than -30 mOsm, c-waves with fast rise-times (cF-wave) were recorded. The peak time of the cS-waves ranged from 4-6 seconds, while that of the cF-waves was approximately 2 seconds. In addition, changes in the pigment granule distribution were also investigated histologically and photometrically. At delta pi of +10 mOsm, the distribution of pigment granules was similar to that in the dark-adapted chick retina. However, when the delta pi was -30 mOsm, the distribution of pigment granules was similar to that in the light-adapted chick retina. Under the control condition at delta pi of +10 mOsm, the pigment granules were distributed mainly within the cell body of the pigment epithelium, ie, in a direction opposite to the passive flow determined by the artificial osmotic gradient. However, after treatment by ouabain, the granules were spread toward the neural retina in the same direction as the passive flow, suggesting passive movement of these granules. These results suggest that a steep osmotic gradient is present within the normal retina and that the gradient is maintained by an ATPase-dependent mechanism. Moreover, the above results suggest that the cS-wave is scotopic and the cF-wave is photopic.

Spectral sensitivity of monochromatic ERG c-wave of chicken under color adaptation.

The spectral sensitivity of the ERG c-wave was studied in the chicken under yellow or red light adaptation of various intensities, ie, 1, 3, 4 and 5 W/m2 at the corneal surface in the former, and 3, 4 and 5 W/m2 in the latter. The peak wavelength of the spectral sensitivity curve of the c-wave amplitude under both yellow and red light adaptations was 520 or 540 nm, being shorter as compared with the peak wavelength (560 nm) under white light adaptation (Fukuda, 1989). Under the yellow or red light adaptation the sensitivity of the c-wave was suppressed at a range of longer wavelength, suggesting a possible isolation of the blue and green cone-driven c-wave responses. The peak (520 or 540 nm) and the shoulder (580 nm) in the spectral sensitivity curves were presumed to be derived from the three cone systems.

Multicenter clinical trial for evaluating methylprednisolone pulse treatment of idiopathic optic neuritis in Japan. Optic Neuritis Treatment Trial Multicenter Cooperative Research Group (ONMRG).

BACKGROUND: A randomized, controlled clinical trial was conducted in 1991 to compare an intravenous megadose of methylprednisolone with a control drug (mecobalamin) for treating acute idiopathic optic neuritis. CASES: Sixty-six cases from 22 clinical centers throughout Japan were examined to evaluate the treatment on visual function parameters, such as visual acuity, visual field, color vision, contrast sensitivity, and critical flicker frequency. OBSERVATIONS: The methylprednisolone pulse treatment group showed faster recovery of visual function, particularly the visual acuity at 1 week (P<.05), Humphrey field analyzer mean deviation at 3 weeks (P<.05), and color vision at 1 week (P<.05). Recovery of contrast sensitivity at several different spatial frequencies was significant in the pulse treatment group at 1 (P<.01), 2 (P<.05), and 4 weeks (P<.05) after the start of treatment. Visual function test results at 12 weeks and 1 year were essentially the same in the two treatment groups. Side effects appeared more frequently in the pulse treatment group than in the control (P<.05). CONCLUSIONS: Pulse treatment does not appear effective for idiopathic optic neuritis even though visual function in the pulse treatment group of this trial recovered more quickly during the initial phase compared to the controls. More effective and specific treatment should be established for optic neuritis.

Baseline features of idiopathic optic neuritis as determined by a multicenter treatment trial in Japan. Optic Neuritis Treatment Trial Multicenter Cooperative Research Group (ONMRG).

BACKGROUND: An optic neuritis treatment trial was conducted at 30 clinical centers in Japan using the same protocol. Patient participation was based on: age range of 14-55 years; acute symptoms indicative of unilateral optic neuritis of unknown or demyelinating origin; visual symptoms of 14-day duration or less; relative afferent pupillary defect in affected eye; and normal or swollen optic disc of affected eye. CASES: Initially, 102 patients qualified for participation; baseline data were obtained for analysis from 70 of these patients. Demographic characteristics of Japanese patients with optic neuritis were clarified and compared with those in a US study. OBSERVATIONS: The incidence of ocular or periocular pain and the presence of periventricular plaques were noted to be lower, and the incidence of disc swelling higher, in the Japanese patients, suggesting racial differences in the characteristics of the disease. Such differences may possibly be related to the lower incidence of multiple sclerosis in Japanese patients. The results of visual function tests were virtually the same in both studies. The nonaffected eyes of more than half the patients showed abnormal mean deviation in Humphrey field analysis, as also noted in the US study. CONCLUSIONS: The baseline clinical features of optic neuritis in the Japanese patients have been defined. Some racial differences in the characteristics of the disease may exist.

Refractive results of post penetrating keratoplasty photorefractive keratectomy.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the safety, effectiveness, and predictability of photorefractive keratectomy (PRK) for severe myopia and astigmatism following penetrating keratoplasty. PATIENTS AND METHODS: PRK was performed on 42 eyes, and 33 eyes were followed up for at least 6 months. RESULTS: Mean preoperative spherical equivalent was -8.29 +/- 4.01 diopters (D), which decreased to -2.96 +/- 3.26 D in manifest refraction at 6 months. Keratometric power reduced from 48.06 +/- 3.32 D preoperatively to 43.97 +/- 3.40 D. Refractive and keratometric astigmatism attained the reduction of 31.0% and 13.56% in average respectively. Twenty three eyes had improved uncorrected visual acuity. CONCLUSION: PRK was effective in reducing post-keratoplasty myopia and astigmatism, but the predictability was not as good as in the non-grafted eye.

[Electrical responses from the inner retina of rats with streptozotocin-induced early diabetes mellitus].

PURPOSE: To evaluate behavior of the scotopic threshold response (STR) and the oscillatory potential (OP) in the electroretinogram (ERG) in streptozotocin (STZ)-induced diabetic rats (DM rats). METHODS: The amplitude of the STR and the OP 3, the implicit time of the STR, and the peak latency of the OP 3 of the DM rats were measured. RESULTS: No significant differences were observed in the mean amplitude or the mean implicit time of the STR between the control and the DM rats. On the other hand, the mean peak latency of OP 3 of the DM rats was significantly prolonged up to 125% of the control rats (p < 0.01), although there were no significant differences between the two groups in the mean OP 3 amplitude. CONCLUSIONS: Although both STR and OP were of inner retinal origin, their behavior was different in DM rats. This result supported some reports describing how dopaminergic amacrine cells generate OP and glycinergic or GABAergic amacrine cells generate STR. In the early stage of diabetic retinopathy, there may be not only minor ischemia but also disorder of neurotransmission of the amacrine cells in the inner retinal layers.