Structural, enzymatic and molecular studies in a series of nonbullous congenital ichthyosiform erythroderma patients.

Causative gene defects have not been demonstrated in the majority of nonbullous congenital ichthyosiform erythroderma (NBCIE) cases. The purpose of this study was to further elucidate the pathogenesis of NBCIE. Immunohistochemical and ultrastructural observations, transglutaminase activity assays and sequencing of TGM1 were performed in five patients from four NBCIE families. Transglutaminase 1 (TGase 1), involucrin and loricrin expression and in situ transglutaminase activity were present in all of the cases. Ultrastructurally, two cases out of five showed incomplete thickening of the cornified cell envelope (CCE) during keratinization and the other three exhibited abnormal lipid droplets in the cornified cells and malformed lamellar granules. No TGM1 mutation was found in any of the four families by direct sequence analysis. NBCIE cases with normal TGase 1 seemed to have two distinct patterns of abnormality, one with abnormal lipid droplets and malformed lamellar granules and the other with defective CCE formation.

Darier's disease restricted to sun-exposed areas.

We report a sporadic case of Darier's disease restricted to sun-exposed areas in a 17-year-old Japanese girl. There are several clinical variants of Darier's disease including unilateral Darier's disease, localized Darier's disease, segmental Darier's disease, and acral Darier's disease, but few cases of Darier's disease restricted to sun-exposed areas have been described in the literature. Although it remains controversial whether UV irradiation can evoke the eruption of Darier's disease or not, cases of Darier's disease restricted to sun-exposed areas like our case may help to further clarify the relationship between Darier's disease, UV irradiation and photo-exacerbation of this autosomal dominant genodermatosis.

Carcinomatous transformation of eccrine syringofibroadenoma.

BACKGROUND: While squamous cell carcinoma and pseudocarcinomatous hyperplasia have been documented as pre-existing lesions in cases of reactive eccrine syringofibroadenoma (ESFA), to the best of our knowledge carcinoma occurring in a solitary ESFA has not yet been reported. We present one such case in a 91-year-old female who had a dome-shaped, reddish tumor on the extensor side of the left forearm. METHODS: We review the histopathological, immunophenotypical and ultrastructural findings of this tumor, including the keratin expression profile. RESULTS: Histopathologically, long, branching, anastomosing, thin and thick strands of small cuboidal epithelial cells were extending from the surface epidermis into the dermis. In the center of the tumor, there were irregular-shaped nests of atypical tumor cells invading downward into the dermis. Ultrastructurally, duct-like lumina lined with cuboidal tumor cells were present in the epithelial cords. From these findings, the present case was diagnosed as solitary eccrine syringofibroadenocarcinoma (ESFAC). Keratin expression studies revealed that cells of the thick strands, except for the luminal and basal cells, were positive for differentiation-specific keratins, keratins 1 and 10, and that cells of the thin strands were positive for keratins 5 and 14. CONCLUSIONS: Histopathological, immunophenotypical and ultrastructural evidence, as well as the pattern of keratin expression, suggest differentiation of the present malignant tumor towards the eccrine dermal duct. This case is the first reported case of ESFAC as far as we know.

Disruption of the suprabasal keratin network by mutation M150T in the helix initiation motif of keratin 10 does not affect cornified cell envelope formation in human epidermis.

Keratin 10 (K10) is known to be tightly bound to the cornified cell envelope (CCE) and this binding is thought to play an important role in enhancing the structural integrity of the cornified cells. Bullous congenital ichthyosiform erythroderma (BCIE) is a genetic disorder of keratinization caused by gene mutations in the conserved sequences of keratin 1 (K1) or K10, which leads to abnormal suprabasal keratin network assembly. In BCIE patients' skin, the keratin network abnormalities make the upper spinous and granular keratinocytes fragile and result in blister formation. However, the exact pathomechanism of the hyperkeratosis seen in BCIE is still unknown. The involvement of the CCE in the pathomechanism of hyperkeratosis in BCIE is controversial. Abnormal CCE assembly may cause hyperkeratosis as reported in cases of lamellar ichthyosis. Binding of K10 to CCE is thought to be a vital connection between the suprabasal keratin filament network and CCE. We hypothesize that abnormal suprabasal keratin assembly caused by either K1 or K10 mutations can disrupt CCE formation, resulting in the hyperkeratosis observed in BCIE. To clarify whether K10 and keratin network defects affect CCE formation in vivo, the ultrastructural and immunohistological features of CCE were studied in the epidermis of two Japanese BCIE patients from two independent families carrying an identical missense mutation M150T in the helix initiation motif of K10. Ultrastructurally, a 15-nm-thick, dense, normal-appearing CCE was formed at the cell periphery of the keratinized epidermal cells. Light and electron microscopic immunolabeling revealed that the major CCE precursor proteins, involucrin and loricrin, were normally distributed and restricted to CCE of the epidermis. Immunofluorescent labeling showed that epidermal TGases, TGase 1, TGase 2 and TGase 3, were expressed normally in the epidermis. These findings suggest that a normal CCE is formed during the process of human epidermal keratinization, even if the suprabasal keratin filament network is disrupted as with this particular K10 mutation, M150T in BCIE.

Formation of cornified cell envelope in human hair follicle development.

BACKGROUND: Cornified cell envelope (CCE) formation is an important step in the final stage of keratinization, in which CCE precursor proteins including involucrin and loricrin are cross-linked by keratinocyte transglutaminases (TGases) to the inner surface of the plasma membrane of cornified cells, while the outer surface is coated with material derived from secreted lamellar granules. OBJECTIVES: Skin samples from human fetuses of a series of estimated gestational age (EGA) (49-163 days) were studied for the prescence of precursor proteins. Methods TGase activity was studied by in situ TGase activity assay, and ultrastructural features of CCE formation were observed at each stage of hair follicle development. We used immunofluorescent labelling to investigate the time and site of expression of CCE precursor proteins involucrin and loricrin, TGases 1, 2 and 3, and a 25-kDa lamellar granule-associated protein (LGP) in developing human hair follicles. RESULTS: In the hair germ (65-84 days EGA) (corresponding to the stages 1-2 of murine hair follicle morphogenesis), only TGase 2 was observed in the entire hair germ, where in situ TGase activity was weakly positive, although thickening of cell membrane was not seen ultrastructurally. In the hair peg (85-104 days EGA) (corresponding to the stage 3 of murine hair follicle morphogenesis), loricrin and TGase 2 were seen in cells of the upper part of the hair peg while TGase 1, 3 and LGP were observed in the inner cells of the hair peg. In situ TGase activity was weakly positive in the upper part and inner cells of the hair peg. In the bulbous hair peg (105-135 days EGA) (corresponding to the stages 4-6 of murine hair follicle morphogenesis) and differentiated lanugo hair follicle (> 135 days EGA) (corresponding to the stages 7-8 of murine hair follicle morphogenesis), immunoreactivities of involucrin, loricrin, TGase 1, 2, 3, in situ TGase activity and LGP were detected in the inner root sheath cells, hair canals and inner cells of the outer root sheath in the region of the isthmus. Ultrastructurally, thickening of cell membrane was already seen in the inner root sheath cells of the bulbous hair peg and electron-dense, thick CCE was observed in the hair cuticle and hair canal of differentiated lanugo hair follicle. CONCLUSIONS: These data indicate that, in terms of CCE formation, certain portions of the developing human hair follicle have already been determined in differentiation of the hair canal and cuticle at the hair peg stage.

Echolocation of multiple targets in 3-d space from a single emission.

Using frequency-modulated echolocation sound, bat can capture a moving target in real three-dimensional (3-D) space. It is impossible to locate multiple targets in 3-D space by using only the delay time between an emission and the resultingechoes received at two points (i.e., two ears). To locate multiple targets in 3-D space requires directional information for each target. The spectrum of the echoes from nearly equidistant targets includes spectral components of both the interference between the echoes and the interference resulting from the physical process of reception at the external ear. The frequency of the spectral notch, which is the frequency corresponding to the minimum of the external ear's transfer function (EEDNF), provides a crucial cue for directional localization. In the model we present, a computational model todiscriminate multiple close targets in 3-D space utilizing echoes evoked by a single emission by distinguishing the interference of echoes from each object and the EEDNF corresponding to each target.

Emx2 directs the development of diencephalon in cooperation with Otx2.

The vertebrate brain is among the most complex biological structures of which the organization remains unclear. Increasing numbers of studies have accumulated on the molecular basis of midbrain/hindbrain development, yet relatively little is known about forebrain organization. Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, we have examined Emx2(-/-)Otx2(+/-) double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2(+/Emx2)). Emx2(-/-)Otx2(+/-) double mutants did not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects were attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure were also present in the double mutants. In contrast, Otx2(+/Emx2) knock-in mutants displayed the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure were specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum.

A model of echolocation of multiple targets in 3D space from a single emission.

Bats, using frequency-modulated echolocation sounds, can capture a moving target in real 3D space. The process by which they are able to accomplish this, however, is not completely understood. This work offers and analyzes a model for description of one mechanism that may play a role in the echolocation process of real bats. This mechanism allows for the localization of targets in 3D space from the echoes produced by a single emission. It is impossible to locate multiple targets in 3D space by using only the delay time between an emission and the resulting echoes received at two points (i.e., two ears). To locate multiple targets in 3D space requires directional information for each target. The frequency of the spectral notch, which is the frequency corresponding to the minimum of the external ear's transfer function, provides a crucial cue for directional localization. The spectrum of the echoes from nearly equidistant targets includes spectral components of both the interference between the echoes and the interference resulting from the physical process of reception at the external ear. Thus, in order to extract the spectral component associated with the external ear, this component must first be distinguished from the spectral components associated with the interference of echoes from nearly equidistant targets. In the model presented, a computation that consists of the deconvolution of the spectrum is used to extract the external-ear-dependent component in the time domain. This model describes one mechanism that can be used to locate multiple targets in 3D space.

The clinical transition between pemphigus foliaceus and pemphigus vulgaris correlates well with the changes in autoantibody profile assessed by an enzyme-linked immunosorbent assay.

BACKGROUND: There are a number of reports of pemphigus with clinical shifting between pemphigus foliaceus (PF) and pemphigus vulgaris (PV). On the other hand, a novel enzyme-linked immunosorbent assay (ELISA) against recombinant baculoproteins of desmoglein 1 (Dsg1) (PF antigen) and Dsg3 (PV antigen) has been established and found to be extremely sensitive and specific. OBJECTIVES: To characterize the change in the antibody profiles in a series of pemphigus cases with mixed features of PF and PV by various methods, including the novel ELISA. Patients/methods Sera were obtained from eight cases undergoing a shift between PF and PV and three cases of coexistent PF and PV. The autoantigens were analysed by ELISA, as well as by immunofluorescence using normal human skin sections and immunoblotting using normal human epidermal extracts. RESULTS: The results of the ELISA, immunofluorescence and immunoblotting studies showed that the transition between PF and PV correlates well with the changes of autoantibodies against either Dsg1 or Dsg3. CONCLUSIONS: The clinical phenotype at each stage is defined by the anti-Dsg antibody profile in the serum of these pemphigus patients showing mixed features of PF and PV. In addition, ELISA using recombinant baculoproteins was particularly useful in distinguishing PF and PV.

Complementary functions of Otx2 and Cripto in initial patterning of mouse epiblast.

The development of the mammalian antero-posterior (A-P) axis is proposed to be established by distinct anterior and posterior signaling centers, anterior visceral endoderm and primitive streak, respectively. Knock-out studies in mice have shown that Otx2 and Cripto have crucial roles in the generation and/or functions of these anterior and posterior centers, respectively. In both Otx2 and Cripto single mutants, the initial formation of the A-P axis takes place in a proximal-distal (P-D) orientation, but subsequent axis rotation fails to occur. To examine the developmental consequences of the lack of these two genes, we have analyzed the Otx2(-/-);Cripto(-/-) double homozygous mutant phenotype. In the double mutants, the expression of the A-P axis markers Cer-l, Lim1, and Wnt3 was not induced, while expression of Fgf8 and T was expanded throughout the epiblast, indicating that the double mutants could not form the A-P axis even in its initial P-D orientation. In addition, the double mutants displayed defects in differentiation of the visceral endoderm overlying the epiblast, as well as in the extraembryonic ectoderm. Furthermore, differentiation of neuroectoderm was accelerated as judged by the reduction of Oct4 expression and emergence of Sox1 and Gbx2 expression in the double mutant epiblast. The resulting ectoderm only displayed characteristics of anterior hindbrain, implicating it as a ground state in the mammalian body plan. Our results indicate that complementary functions of Otx2 and Cripto are essential for initial patterning of the A-P axis in the mouse embryo.

Glutamate release via NO production evoked by NMDA in the NTS enhances hypotension and bradycardia in vivo.

Nitric oxide (NO) in the nucleus tractus solitarii (NTS) plays an important role in regulating sympathetic nerve activity. The aims of this study were to determine whether the activation of N-methyl-D-aspartate (NMDA) receptors in the NTS facilitates the release of L-glutamate (Glu) via NO production, and, if so, to determine whether this mechanism is involved in the depressor and bradycardic responses evoked by NMDA. We measured the production of NO in the NTS as NO2- and NO3- (NO(x)) or Glu levels by in vivo microdialysis before, during, and after infusion of NMDA in anesthetized rats. We also examined effects of N(omega)-nitro-L-arginine methyl ester (L-NAME) on the changes in these levels. NMDA elicited depressor and bradycardic responses and increased the levels of NO(x) and Glu. L-NAME abolished the increases in the levels of NO(x) and Glu and attenuated cardiovascular responses evoked by NMDA. These results suggest that NMDA receptor activation in the NTS induces Glu release through NO synthesis and that Glu released via NO enhances depressor and bradycardic responses.

Synchronized disappearance of serum HCV-RNA, anti-U1 RNP, anti-La/SS-B, and anti-Scl-70 in a patient with chronic hepatitis.

The authors report a rare case of chronic hepatitis in whom normalization of serum aminotransferases was associated with disappearance of serum hepatitic C virus (HCV)-ribonucleic acid (RNA), anti-U1 RNP, anti-La/SS-B, and anti-Scl-70 antibodies without treatment of interferon or corticosteroids. A 27-year-old Japanese woman was diagnosed with chronic hepatitis C, with positive anti-nuclear antibody, anti-U1 RNP, anti-La/SS-B, and anti-Scl-70 antibodies. Histopathologic examination of a liver biopsy specimen showed a periportal interface hepatitis with a predominantly lymphoplasmacytic necroinflammatory infiltrate and lobular hepatitis. After two-year treatment with ursodeoxycholic acid (UDCA), serum aminotransferases normalized and serum HCV-RNA, anti-U1 RNP, anti-La/SS-B, and anti-Scl-70 antibodies disappeared. It was unclear whether disappearance of HCV-RNA was spontaneous, due to some immunomodulating effects of UDCA, or other unknown mechanism, but host immune response may be associated with HCV elimination.

Changes in titers of antimitochondrial and antinuclear antibodies during the course of primary biliary cirrhosis.

A case of primary biliary cirrhosis (PBC) in whom a complete biochemical (serum bilirubin, transaminases and alkaline phosphatase) remission was noted after combination treatment with ursodeoxycholic acid (UDCA) and corticosteroid is reported. The antimitochondrial antibody (AMA) detected by indirect immunofluorescence was initially positive, and the antinuclear antibody (ANA) was negative, but these two antibodies subsequently fluctuated independently (AMA-positive/ANA-negative, AMA-negative/ANA-negative, AMA-negative/ANA-positive, AMA-positive/ANA-positive, and again AMA-negative/ANA-positive) in spite of a lack of histopathological improvement in the liver after treatment. The clinical presentation in our case suggests that in some cases the diagnosis of PBC or so-called autoimmune cholangitis (AIC) might depend on the 'phase' of the same disease. Our results also suggest that detailed immunoreactive profiles against 2-oxo-acid dehydrogenase complex (2-OADC) enzymes by using immunoblotting, together with a serial histological examination, should provide more precise information for a diagnosis of PBC.

Clinical features and treatment of hepatocellular carcinoma in eight patients older than eighty years of age.

BACKGROUND/AIMS: Most-hepatocellular carcinoma patients are between 40 and 60 years of age, but an increasing number of elderly patients with hepatocellular carcinoma is expected in the future because of the increase in life expectancy seen in many countries. Since elderly patients have a high incidence of comorbid illnesses, it should be useful to examine the clinical features of these patients to select the optimal management strategy for hepatocellular carcinoma. METHODOLOGY: A retrospective review of 111 patients with hepatocellular carcinoma was undertaken to examine the clinical features of 8 patients older than 80 years of age. RESULTS: In the 111 patients with hepatocellular carcinoma, the ratio of males to females was 81:30 and the peak incidence of hepatocellular carcinoma was noted in the seventh and eighth decades in males and females, respectively. Of these, 21 (19%) were type "B" [seropositive for hepatitis B surface antigen (HBsAg) and seronegative for antibody to the hepatitis C virus (anti-HCV)], 69 (62%) were type "C" (seronegative for HBsAg and seropositive for anti-HCV), 3 (3%) were type "B + C" (seropositive for both HBsAg and anti-HCV), and 18 (16%) were type "non-B non-C" (seronegative for both HBsAg and anti-HCV). The peak incidences of type "B" were in the sixth decade, whereas those of type "C" were in the seventh decade in both males and females. Patients with "non-B non-C" were common in their seventies. Of the 111 patients, 6 (5 males and 1 female) were older than 80 years at the time of diagnosis and 2 females became 80 years old during the course of follow-up of hepatocellular carcinoma. All but one of these patients were anti-HCV-positive, stage and clinical stage I or II according to the criteria defined by the Liver Cancer Study Group of Japan, and underwent transcatheter arterial embolization and/or transcatheter arterial infusion chemotherapy. Transcatheter arterial embolization/transcatheter arterial infusion or percutaneous ethanol injection therapy was well tolerated in these patients, and the outcome of these patients was good. However, concomitant underlying diseases other than liver diseases made it impossible or difficult to apply an aggressive management protocol for hepatocellular carcinoma in some patients. CONCLUSIONS: Our results suggest that the overall treatment of hepatocellular carcinoma in the elderly should be similar to that in younger patients, but may be restricted by the concomitant underlying diseases specific to advanced age.

Serial changes in enzyme inhibitory antibody to pyruvate dehydrogenase complex during the course of primary biliary cirrhosis.

To assess the usefulness of enzyme inhibition assay for the diagnosis of primary biliary cirrhosis (PBC), we determined the serial changes in enzymatic inhibitory antibody to pyruvate dehydrogenase complex (PDC) in patients with PBC, and compared the results to those of immunofluorescence and immunoblotting. Forty-nine sera from 19 patients with PBC who were followed-up for at least 16 months were tested for antimitochondrial antibodies (AMA) by indirect immunofluorescence, immunoblotting on bovine heart mitochondria, and enzyme inhibition assay using commercially available TRACE Enzymatic Mitochondrial Antibody (M2) Assay (EMA) kit. Of the 49 sera, 39 (80%), 35 (71%), 38 (78%), 31 (63%), and 36 (73%) were positive for AMA by immunofluorescence, for immunoglobulin G (IgG), IgM, and IgA class antibody against E2 subunit of PDC (PDC-E2) by immunoblotting, and for enzymatic inhibitory antibody to PDC by EMA, respectively. AMA titers determined by immunofluorescence did not change in 9 patients (47%), increased in 4 (21%), decreased in 3 (16%), and fluctuated in 3 (16%) during follow-up. The number of anti-M2 bands by immunoblotting did not change in 9 (47%), increased in 6 (32%), decreased in 2 (11%), and fluctuated in 2 (11%). Units of PDC activity by EMA did not change markedly in 16 (84%), increased in 2 (11%), and fluctuated in 1 (5%). Positive EMA results were common in cases with high levels of serum alkaline phosphatase and IgM, and the units of PDC activity by EMA correlated significantly and inversely with AMA titers by immunofluorescence, and serum reactivity to PDC-E2 by immunoblotting, respectively. There was no correlation between serial changes in biochemical data and units of PDC activity by EMA. In three patients who showed a decrease in AMA titers, AMA titers correlated more with EMA results than immunoblotting. Moreover, in a patient with fluctuating AMA titers, the units of PDC activity by EMA paralleled AMA titers. Our results suggest that EMA is useful for the diagnosis of AMA-positive PBC, and also could be used for monitoring the disease course in PBC.

A new murine zinc finger gene, Opr.

Here we report a novel murine zinc-finger gene, Opr, belonging to the opa/Zic family. Opr is expressed in the entire embryonic ectoderm before gastrulation, but gradually restricted to the anterior part from the mid to late streak stage. At the beginning of neural induction, Opr is expressed throughout the anterior neural plate, but is soon restricted to the neural ridge. After neural tube closure, its expression is maintained in the dorsal part of the neural tube, except for the roof of the telencephalon. Opr is also expressed in somites and limbs.

Visceral endoderm mediates forebrain development by suppressing posteriorizing signals.

The anterior visceral endoderm (AVE) has attracted recent attention as a critical player in mouse forebrain development and has been proposed to act as "head organizer" in mammals. However, the precise role of the AVE in induction and patterning of the anterior neuroectoderm is not yet known. Here we identified a 5'-flanking region of the mouse Otx2 gene (VEcis) that governs the transgene expression in the visceral endoderm. In transgenic embryos, VEcis-active cells were found in the distal visceral endoderm at 5.5 days postcoitus (dpc), had begun to move anteriorly at 5.75 dpc, and then became restricted to the AVE prior to gastrulation. The VEcis-active visceral endoderm cells exhibited ectodermal morphology distinct from that of the other endoderm cells and consisted of two cell layers at 5.75 dpc. In the Otx2(-/-) background, the VEcis-active endoderm cells remained distal even at 6.5 dpc when a primitive streak was formed; anterior definitive endoderm was not formed nor were any markers of anterior neuroectoderm ever induced. The Otx2 cDNA transgene under the control of the VEcis restored these Otx2(-/-) defects, demonstrating that Otx2 is essential to the anterior movement of distal visceral endoderm cells. In germ-layer explant assays between ectoderm and visceral endoderm, the AVE did not induce anterior neuroectoderm markers, but instead suppressed posterior markers in the ectoderm; Otx2(-/-) visceral endoderm lacked this activity. Thus Otx2 is also essential for the AVE to repress the posterior character. These results suggest that distal visceral endoderm cells move to the future anterior side to generate a prospective forebrain territory indirectly, by preventing posteriorizing signals.