Development of eosinophilic granulomatosis with polyangiitis during the clinical course of microscopic polyangiitis: A case report.

RATIONALE: Eosinophilic granulomatosis with polyangiitis (EGPA) is belongs to the antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) subgroups. EGPA, unlike other subgroups of AAV, including microscopic polyangiitis (MPA) and granulomatosis with polyangiitis, has the unique feature that both ANCA and eosinophilic inflammation are involved in its pathogenesis. Although AAV often relapses, there are currently no reports of EGPA developing during other subgroups of AAV. Herein, we document a case of EGPA that developed during the clinical course of MPA. PATIENT CONCERNS: A 61-year-old Japanese woman was diagnosed with MPA based on interstitial lung disease and myeloperoxidase-ANCA positivity. After starting immunosuppression therapy, including prednisolone and tacrolimus, she was expected to achieve clinical remission. Nonetheless, she occasionally experienced MPA relapse, which required an increased prednisolone dose, rituximab, intravenous cyclophosphamide, and plasma exchange. Three years after MPA onset, she developed renal amyloidosis; thus, subcutaneous tocilizumab was added to her regimen. Following clinical remission, the administration interval of her subcutaneous tocilizumab therapy was extended and immunosuppressants were discontinued. She then developed bronchial asthma and mild eosinophilia (eosinophilic count: ~1000/µL). Further, a year later, she underwent total hip replacement using a titanium implant. Subsequently, she developed abnormal sensation in both hands, numbness, and muscle weakness, as well as palpable purpura and massive eosinophilia (eosinophilic count: ~8500/µL). DIAGNOSIS: We diagnosed the patient with EGPA based on 5 items (asthma, multiple mononeuropathies, sinus abnormality, and extravascular eosinophils) of the 1990 American College of Rheumatology classification criteria. INTERVENTIONS: We administered 400 mg/kg intravenous immunoglobulin for 5 consecutive days, 300 mg mepolizumab subcutaneously every 4 weeks, and 40 mg/day prednisolone following pulsed methylprednisolone therapy (1000 mg/day for 3 consecutive days). OUTCOMES: After these treatments, the patient's symptoms improved, and eosinophilic count and inflammatory markers declined. LESSONS: The present case suggests that EGPA can be induced by the development of eosinophilic inflammation in other subgroups of AAV.

Preferential freezing avoidance localised in anthers and embryo sacs in wintering Daphne kamtschatica var. jezoensis flower buds visualised by magnetic resonance imaging.

To explore diversity in cold hardiness mechanisms, high resolution magnetic resonance imaging (MRI) was used to visualise freezing behaviours in wintering Daphne kamtschatica var. jezoensis flower buds, which have naked florets and no bud scales. MRI images showed that anthers remained stably supercooled to the range from -14 to -21°C or lower while most other tissues froze by -7°C. Freezing of some anthers detected in MRI images between -14 and -21°C corresponded with numerous low temperature exotherms and also with the 'all-or-nothing' type of anther injuries. In ovules/pistils, only embryo sacs remained supercooled at -7°C or lower, but slowly dehydrated during further cooling. Cryomicroscopic observation revealed ice formation in the cavities of calyx tubes and pistils but detected no ice in embryo sacs or in anthers. The distribution of ice nucleation activity in floral tissues corroborated the tissue freezing behaviours. Filaments likely work as the ice blocking barrier that prevents ice intrusion from extracellularly frozen calyx tubes to connecting unfrozen anthers. Unique freezing behaviours were demonstrated in Daphne flower buds: preferential freezing avoidance in male and female gametophytes and their surrounding tissues (by stable supercooling in anthers and by supercooling with slow dehydration in embryo sacs) while the remaining tissues tolerate extracellular freezing.

[Preservation of the Spinal Perfusion Artery during Descending Thoracic and Thoracoabdominal Aortic Surgery].

This is a 3-case report of successful descending thoracic and thoracoabdominal aortic surgery by preserving the spinal perfusion artery (SPA) identified preoperatively. In Case 1, an 85-year-old woman, computed tomography (CT) showed Crawford type Ⅴ thoracoabdominal aortic aneurysm (TAAA:60 mm) and a SPA originated from L2. In Case 2, a 76-year-old man, CT revealed type Ⅳ TAAA( 58 mm) and a SPA originated from Th11. In Case 3, a 74-year-old man, CT detected an infectious pseudoaneurysm(44 mm) in the descending thoracic aorta with 2 SPAs originating from Th10 and L2. The ranges of graft replacement were Th7-Th12, Th12-L4, and Th8-Th10, respectively, while preserving all SPAs. All patients recovered well without postoperative neurological deficits. Although the protective effect of the SPA preservation against the spinal cord ischemia is still controversial, preoperative identification of the SPA was useful for planning a surgical strategy for descending thoracic and thoracoabdominal aortic repair surgery.

Freezing behaviours in wintering Cornus florida flower bud tissues revisited using MRI.

How plant tissues control their water behaviours (phase and movement) under subfreezing temperatures through adaptative strategies (freezing behaviours) is important for their survival. However, the fine details of freezing behaviours in complex organs and their regulation mechanisms are poorly understood, and non-invasive visualization/analysis is required. The localization/density of unfrozen water in wintering Cornus florida flower buds at subfreezing temperatures was visualized with high-resolution magnetic resonance imaging (MRI). This allowed tissue-specific freezing behaviours to be determined. MRI images revealed that individual anthers and ovules remained stably supercooled to -14 to -21 °C or lower. The signal from other floral tissues decreased during cooling to -7 °C, which likely indicates their extracellular freezing. Microscopic observation and differential thermal analyses revealed that the abrupt breakdown of supercooled individual ovules and anthers resulted in their all-or-nothing type of injuries. The distribution of ice nucleation activity in flower buds determined using a test tube-based assay corroborated which tissues primarily froze. MRI is a powerful tool for non-invasively visualizing unfrozen tissues. Freezing events and/or dehydration events can be located by digital comparison of MRI images acquired at different temperatures. Only anthers and ovules preferentially remaining unfrozen are a novel freezing behaviour in flower buds. Physicochemical and biological mechanisms/implications are discussed.

Bone pattern formation in mouse limbs after amputation at the forearm level.

BACKGROUND: Although urodela amphibians completely regenerate amputated limbs, the potency of limb regeneration in mammalians is very low. In mice and humans, a digit tip regenerates, but no regeneration beyond the digit joint occurs. In this article, we describe the induction of bone formation by BMPs in the forearm, a more proximal region, of neonatal mice. Distal halves of the radius and ulna were removed from limbs amputated at the wrist level in advance, and gelatin rods containing BMPs were grafted in the space of the forearm. BMP-7 and BMP-2 were effective. RESULTS: The induction of bone formation depended on the amount of BMP-7 in grafted gelatin gels. BMP-7 at over 320 ng/limb induced long bones within 10 days. Cartilage formation was always observed before the bone formation as in the case of normal limb development. The origin of cells responsive to BMP-7 is not clear, but the formation of large bone tissues in a relatively short time suggests a wide distribution of precursor cells in the limbs. Grafting of dye-stained mesenchymal tissue fragments suggested that mesenchymal tissues other than the dermis, bone, and joint cartilage are responsive to BMP-7 and form cartilage/bone. Two long bones induced were arranged in parallel fashion in many cases like the radius and ulna. An agonist of hedgehog promoted the formation of bones in the presence of BMP-7. Furthermore, bone elements were fused to stump bones to form a single bone. CONCLUSIONS: The results suggest the potency of bone pattern regeneration in the amputated forearm of a neonatal mouse.

Functional analysis of chick heparan sulfate 6-O-sulfotransferases in limb bud development.

Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O-sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O-sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS-6-O-sulfotransferases (HS6ST-1 and HS6ST-2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf-8 and Fgf-10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST-2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf-8 and Fgf-10 expressions than that achieved with HS6ST-1 RNAi. HS6ST-1 RNAi and HS6ST-2 RNAi caused a significant but distinct reduction in the levels of different 6-O-sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6-O-sulfation of HS play essential roles in chick limb bud development.

Effects of activation of hedgehog signaling on patterning, growth, and differentiation in Xenopus froglet limb regeneration.

Regenerating limbs of urodele amphibians and Xenopus tadpole are reconstructed along proximal-distal, anterior-posterior (AP), and dorsal-ventral axes. In contrast, a regenerated limb of the Xenopus froglet does not have digits, and only a simple cartilaginous structure referred to as a "spike" is formed. This suggests that repatterning along the AP axis is absent in the froglet blastema. Previous studies have shown that Shh and its target genes are not expressed in the froglet blastema. In this study, we activated Hedgehog signaling in the froglet blastema and found that target genes of Shh were inducible in the mesenchyme of limb blastema. Furthermore, we found that activation of the signaling had effects on blastema cell proliferation and chondrogenesis and resulted in the formation of multiple cartilaginous structures. These findings indicate that activation of signaling that is absent in the froglet blastema is effective for improvement of limb regeneration ability in the Xenopus froglet.

Competent stripes for diverse positions of limbs/fins in gnathostome embryos.

Every vertebrate species has its own unique morphology adapted to a particular lifestyle and habitat. Limbs and fins are strikingly diversified in size, shape, and position along the body axis. This diversity in morphology suggests the existence of a variety of embryonic developmental programs. However, comparisons of various embryos suggest common mechanisms underlying limb/fin formation. Here, we report the existence of continuous stripes of competency for appendage formation along the dorsal midline and the lateral trunk of all of the major jawed vertebrate (gnathostome) groups. We also show that the developing fin buds of cartilaginous fish share a mechanism of anterior-posterior axis formation as well as an shh (sonic hedgehog) expression domain in the posterior bud. We hypothesize a continuous distribution of competent stripes that represents the common developmental program at the root of appendage formation in gnathostomes. This schema would have permitted subsequent divergence into various levels of limbs/fins in each animal group.

The autopod: its formation during limb development.

The autopod, including the mesopodium and the acropodium, is the most distal part of the tetrapod limb, and developmental mechanisms of autopod formation serve as a model system of pattern formation during development. Cartilage rudiments of the autopod develop after proximal elements have differentiated. The autopod region is marked by a change in the expression of two homeobox genes: future autopod cells are first Hoxa11/Hoxa13-double-positive and then Hoxa13-single-positive. The change in expression of these Hox genes is controlled by upstream mechanisms, including the retinoic acid pathway, and the expression of Hoxa13 is connected to downstream mechanisms, including the autopod-specific cell surface property mediated by molecules, including cadherins and ephrins/Ephs, for cell-to-cell communication and recognition. Comparative analyses of the expression of Hox genes in fish fins and tetrapod limb buds support the notion on the origin of the autopod in vertebrates. This review will focus on the cellular and molecular regulation of the formation of the autopod during development and evolutionary developmental aspects of the origin of the autopod.

Molecular characterization of axonemal proteins and signaling molecules responsible for chemoattractant-induced sperm activation in Ciona intestinalis.

Spermatozoa undergo dramatic physiological changes at fertilization. In the ascidian Ciona intestinalis, an egg-derived substance named SAAF induces both sperm activation and chemotaxis to the egg. To elucidate the molecular mechanism underlying these phenomena, whole sperm proteins before and after SAAF-treatment were analyzed by two-dimensional gel electrophoresis. By comparison of spot patterns before and after activation, we found twelve proteins that changed the isoelectric points. Seven proteins were shown to be axonemal proteins and others were suggested to be non-axonemal components. Analysis of these proteins by MS-based proteomic system revealed that components of several substructures of the axonemes underwent the changes in isoelectric point at sperm activation, including WD-repeat intermediate chains of outer and inner arm dyneins and a radial spoke protein LRR37, as well as novel axonemal proteins with armadillo repeats or SMC domain. Molecules for cell signaling such as 14-3-3 proteins, Skp1 and VCP/p97 also showed isoelectric changes at sperm activation. These results show a comprehensive feature for signaling mechanism of the activation of spermatozoa at fertilization and also shed new lights on the regulation of ciliary and flagellar movements.

Correlation between Shh expression and DNA methylation status of the limb-specific Shh enhancer region during limb regeneration in amphibians.

The Xenopus adult limb has very limited regeneration ability, and only a simple cartilaginous spike structure without digits is formed after limb amputation. We found that expression of Shh and its downstream genes is absent from the regenerating blastema of the Xenopus froglet limb. Moreover, we found that a limb enhancer region of the Shh gene is highly methylated in the froglet, although the sequence is hypomethylated in the Xenopus tadpole, which has complete limb regeneration ability. These findings, together with the fact that the promoter region of Shh is hardly methylated in Xenopus, suggest that regenerative failure (deficiency in repatterning) in the Xenopus adult limb is associated with methylation status of the enhancer region of Shh and that a target-specific epigenetic regulation is involved in gene re-activation for repatterning during the Xenopus limb regeneration process. Because the methylation level of the enhancer region was low in other amphibians that have Shh expression in the blastemas, a low methylation status may be the basic condition under which transcriptional regulation of Shh expression can progress during the limb regeneration process. These findings provide the first evidence for a relationship between epigenetic regulation and pattern formation during organ regeneration in vertebrates.

Essential role of heparan sulfate 2-O-sulfotransferase in chick limb bud patterning and development.

The interactions of heparan sulfate (HS) with heparin-binding growth factors, such as fibroblast growth factors (FGFs), depend greatly on the chain structures. O-Sulfations at various positions on the chain are major factors determining HS structure; therefore, O-sulfation patterns may play a crucial role in controlling the developmental and morphogenetic processes of various tissues and organs by spatiotemporally regulating the activities of heparin-binding growth factors. In a previous study, we found that HS-2-O-sulfotransferase is strongly expressed throughout the mesoderm of chick limb buds during the early stages of development. Here we show that inhibition of HS-2-O-sulfotransferase in the prospective limb region by small inhibitory RNA resulted in the truncation of limb buds and reduced Fgf-8 expression in the apical ectodermal ridge. The treatment also reduced Fgf-10 expression in the mesenchyme. Moreover 2-O-sulfated HS, normally abundant in the basement membranes and mesoderm under ectoderm in limb buds, was significantly reduced in the treated buds. Phosphorylation levels of ERK and Akt were up-regulated in such truncated buds. Thus, we have shown for the first time that 2-O-sulfation of HS is essential for the FGF signaling required for limb bud development and outgrowth.

Regeneration potency of mouse limbs.

Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit-like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP-7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future.

Transgenic Xenopus with prx1 limb enhancer reveals crucial contribution of MEK/ERK and PI3K/AKT pathways in blastema formation during limb regeneration.

Understanding the mechanisms that control amphibian limb regeneration should allow us to decipher the critical differences between amphibians and humans, which have the limited ability of organ regeneration. However, many issues at the cellular and molecular levels still remain unresolved. We have generated a transgenic Xenopus laevis line that expresses green fluorescent protein (GFP) under the control of mouse prx1 limb enhancer, which directs reporter gene expression in limb mesenchyme in mice, and found that GFP accumulated in blastemal mesenchymal cells of the transgenic froglets after limb amputation. Thus, this transgenic line should provide a new approach to gain insights into the cellular dynamics and signaling pathways involved in limb blastema formation. We have also developed a culture system for forelimb explants of froglets and found that treatment with inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase 1/2 (MEK1/2) and phosphatidylinositol 3-kinase (PI3K) repressed GFP expression. These effects were partially reversible, and down-regulation of GFP was associated with inhibition of cell-cycle progression and induction of ectopic apoptosis. In addition, we found that ERK1/2 and AKT, downstream mediators of MEK1/2 and PI3K pathways, were activated in amputated forelimb stumps. These results demonstrate that MEK/ERK and PI3K/AKT pathways regulate limb blastema formation in the X. laevis froglet.

Function of FGF signaling in the developmental process of the median fin fold in zebrafish.

Median fins, unpaired appendages in fish, are fundamental locomotory organs that are believed to have evolved before paired lateral appendages in vertebrates. However, the early process of median fin development remains largely unknown. We investigated the early development of the median fin fold, a rudiment of median fins, and report here the process in zebrafish embryos and the function of FGF signaling in the process. Using expressions of three genes, dlx5a, sp9 and fgf24, as markers of different phases of fold development, our findings suggest that the early process of median fin fold development can be divided into two steps, specification of the median fin fold territory and construction of the fold structure. Both loss-of-function and gain-of-function assays revealed that FGF signaling plays roles in each step, suggesting a common mechanism for the development of median appendages and paired lateral appendages.

Characterization of Xenopus digits and regenerated limbs of the froglet.

Xenopus has 4 and 5 digits in a forelimb and hindlimb, respectively. It is thought that their limbs and digits develop in Xenopus by mechanisms that are almost conserved from amphibians to higher vertebrates. This is supported by some molecular evidence. The 5'hoxd genes are convenient marker genes for characterizing digits in the chick and mouse. The anteriormost digit is characterized by being hoxd13-positive and hoxd12 (hoxd11)-negative in the chick and mouse. In this study, we revealed that the anteriormost digit of the Xenopus forelimb is hoxd13-positive and hoxd11-positive, that is, a more posterior character than digit I. The order of formation of digit cartilages also suggested that Xenopus forelimb digit identity is II to V, not I to IV. We have also been interested in the relationship between digit identity and shh. The anteriormost digit develops in a shh-independent way. A limb treated with cyclopamine (a shh inhibitor) has a gene expression pattern (hoxd11-negative) similar to that in shh-deficient mice, suggesting that a hindlimb treated with cyclopamine has a digit I character. However, a Xenopus froglet regenerate (spike), which lacks shh expression during its regeneration process, does not have such an expression pattern, being hoxd11-positive. We investigated hoxd11 transcriptions in blastemas that formed in the anteriormost and posteriormost digits, and we found that the blastemas have different hoxd11 expression levels. These findings suggest that the froglet limb blastema does not have a mere digit I character in spite of shh defectiveness and that the froglet limb blastema recognizes its positional differences along the anterior-posterior axis.

Molecular characterization of Ciona sperm outer arm dynein reveals multiple components related to outer arm docking complex protein 2.

Using proteomic and immunochemical techniques, we have identified the light and intermediate chains (IC) of outer arm dynein from sperm axonemes of the ascidian Ciona intestinalis. Ciona outer arm dynein contains six light chains (LC) including a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock and two components related to Chlamydomonas LC8. No LC with thioredoxin domains is included in Ciona outer arm dynein. Among the five ICs in Ciona, three are orthologs of those in sea urchin dynein: two are WD-repeat proteins and the third one, unique to metazoan sperm flagella, contains both thioredoxin and nucleoside diphosphate kinase modules. The remaining two Ciona ICs have extensive coiled coil structure and show sequence similarity to outer arm dynein docking complex protein 2 (DC2) that was first identified in Chlamydomonas flagella. We recently identified a third DC2-like protein with coiled coil structure, Ci-Axp66.0 that is also associated in substoichiometric amounts with Ciona outer arm dynein. In addition, Oda5p, a component of an additional complex required for assembly of outer arm dynein in Chlamydomonas flagella, also groups with this family of DC2-like proteins. Thus, the assembly of outer arm dynein onto doublet microtubules involves multiple coiled-coil proteins related to DC2.

Analysis of scleraxis and dermo-1 genes in a regenerating limb of Xenopus laevis.

Xenopus laevis larvae can regenerate an exact replica of the missing part of a limb after amputation at an early limb bud stage. However, this regenerative capacity gradually decreases during metamorphosis, and a froglet is only able to regenerate hypomorphic cartilage, resulting in a spike-like structure (spike). It has been reported that the spike has tissue deformities, e.g., a muscleless structure. However, our previous study demonstrated that the muscleless feature of the spike can be improved. The existence of other kinds of tissue, such as tendon, has not been clarified. In this study, we focused on the tendon and dermis, and we isolated the scleraxis and dermo-1 genes, which are known to be marker genes for the tendon and dermis, respectively. The expressions of these genes were investigated in both the developmental and regenerating processes of a Xenopus limb. Although muscle was needed to maintain scleraxis expression, scleraxis transcription was detectable in the muscleless spike. Additionally, although grafting of matured skin, including dermal tissue, inhibited limb regeneration, the expression of dermo-1, a dermal marker gene, was detected from the early stage of the froglet blastema. These results indicate that tendon precursor cells and dermal cells exist in the regenerating froglet blastema. Our results support the idea that spike formation in postmetamorphic Xenopus limbs is epimorphic regeneration.

Limb regeneration in Xenopus laevis froglet.

Limb regeneration in amphibians is a representative process of epimorphosis. This type of organ regeneration, in which a mass of undifferentiated cells referred to as the "blastema" proliferate to restore the lost part of the amputated organ, is distinct from morphallaxis as observed, for instance, in Hydra, in which rearrangement of pre-existing cells and tissues mainly contribute to regeneration. In contrast to complete limb regeneration in urodele amphibians, limb regeneration in Xenopus, an anuran amphibian, is restricted. In this review of some aspects regarding adult limb regeneration in Xenopus laevis, we suggest that limb regeneration in adult Xenopus, which is pattern/tissue deficient, also represents epimorphosis.