Miyuki Kinebuchi

NKT cells in the rat: organ-specific distribution of NK T cells expressing distinct V alpha 14 chains.

Rat invariant TCR α-chains and NKT cells were investigated to clarify whether CD1d-mediated recognition by NKT cells is conserved further in evolution. Rats had multiple-copies of TRAV14 genes, which can be categorized into two types according to the diversity accumulated in the CDR2 region. Rats retained invariant TCRα forms with the homogeneous junctional region similar to mouse invariant TRAV14-J281. The proportion of invariant TCR among Vα14+ clones was 12.9% in the thymus and increased in the periphery, 31% in the spleen and 95% in hepatic sinusoidal cells. The invariant TRAV14-J281 was expressed by liver sinusoidal and splenic NKT cells with CD8, CD44high, and TCR Vβ8. Type 1 invariant TCRα was expressed more frequently in hepatic lymphocytes, while type 2 invariant TCRα was expressed predominantly in the spleen. Both types of cells cytolyzed to and were stimulated to proliferate by CD1d-expressing cells in a CD1d-restricted manner. These results suggested that rat NKT cells bearing distinct Vα14 chains are distributed in a tissue-specific pattern. NKT cell populations in rats were more variable than those in mice, indicating that they play novel roles in nature. The implication of the molecular interaction between the structurally diverse invariant TCRα and CD1d/ligand complex in different organs is discussed.

The Signaling Adaptor Protein CD3ζ Is a Negative Regulator of Dendrite Development in Young Neurons

A novel idea is emergxsing that a large molecular repertoire is common to the nervous and immune systems, which might reflect the existence of novel neuronal functions for immune molecules in the brain. Here, we show that the transmembrane adaptor signaling protein CD3zeta, first described in the immune system, has a previously uncharacterized role in regulating neuronal development. Biochemical and immunohistochemical analyses of the rat brain and cultured neurons showed that CD3zeta is mainly expressed in neurons. Distribution of CD3zeta in developing cultured hippocampal neurons, as determined by immunofluorescence, indicates that CD3zeta is preferentially associated with the somatodendritic compartment as soon as the dendrites initiate their differentiation. At this stage, CD3zeta was selectively concentrated at dendritic filopodia and growth cones, actin-rich structures involved in neurite growth and patterning. siRNA-mediated knockdown of CD3zeta in cultured neurons or overexpression of a loss-of-function CD3zeta mutant lacking the tyrosine phosphorylation sites in the immunoreceptor tyrosine-based activation motifs (ITAMs) increased dendritic arborization. Conversely, activation of endogenous CD3zeta by a CD3zeta antibody reduced the size of the dendritic arbor. Altogether, our findings reveal a novel role for CD3zeta in the nervous system, suggesting its contribution to dendrite development through ITAM-based mechanisms.

Contribution of Va24+Vb11+ natural killer T cells in Wilsonian hepatitis

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. There is no evidence that the WD patients immune system attacks copper accumulated hepatocytes. Here we describe that the frequency and absolute number of Vα24+Vβ11+ natural killer T (NKT) cells were significantly increased in 3 cases of WD, whereas those of CD3+CD161+ NKT cells were within the normal range. Patients no. 1 and 2 had a presymptomatic form of WD. Their tissue specimens showed pathological changes of mild degeneration of hepatocytes with a few infiltrating mononuclear cells and a low degree of fatty change. Patient no. 3 displayed fulminant hepatitis with Coombs‐negative haemolytic anaemia. The tissue specimens of patient no. 3 showed macronodular cirrhosis with thick fibrosis, inflammatory infiltrates and spotty necrosis. Human Vα24+Vβ11+ NKT cells are almost equal to CD1d‐restricted NKT cells. Therefore we investigated CD1d‐restricted NKT cells in the LEC rat as an animal model of WD. In LEC rats before hepatitis onset, the number and phenotype of liver NKT cells were normal. At about 4 months of age all LEC rats developed acute hepatitis accompanied by acute jaundice, and CD161high NKT cells developed in their livers. CD161highαβTCRbright NKT cells developed in some of them. Their hepatitis was severe. CD161highαβTCRbright NKT cells expressed an invariant rat Vα14‐Jα281 chain, which is CD1d‐restricted. Furthermore, liver lymphocytes in the acute jaundiced LEC rats with CD161highαβTCRbright NKT cells had significant and CD1d‐specific cytotoxic activity.

RT1.P, rat class Ib genes related to mouse TL: evidence that CD1 molecules but not authentic TL antigens are expressed by rat thymus

Abstract CD1 and TL were once thought to be genetic homologues because of their thymus-specific expression. We investigated their equivalents in the rat to clarify whether their structure and pattern of expression are conserved in rodents. Two rat class Ib genes, containing 3′ sequences very similar to mouse TL, were identified and designated RT1.P. Neither of them, however, can encode ordinary class I molecules due to the accumulation of harmful mutations in the 5′ regions that are unique to RT1.P, while the 3′TL-like regions still retain protein-coding capacity. Comparison of the structural organization of three types of TL family genes, which include mouse T3/T18-encoding TL antigens, mouse T1/T16, and rat RT1.P1/P2 pseudogenes, revealed the presence of a clear demarcation between the type-specific and TL-specific sequences at intron 3. This finding suggests that recombination plays an important role in creating the TL family genes in rodents. Characteristic features of TL, such as a low level of polymorphism and linkage to the major histocompatibility complex, were also observed in the rat. On the other hand, rat CD1 molecules were expressed at a high level on the surface of thymocytes. Absence of authentic TL antigens and thymic expression of CD1d molecules in the rat suggest the plasticity and conservation of class Ib genes in rodent evolution. Functions of TL may be substituted with CD1 or other class Ib molecules expressed by rat thymus.

Hemolytic crisis with fulminant hepatic failure in Wilson disease without consanguinity

Departments of 1 Pediatrics and 2 Ophthalmology, Hirosaki City Hospital, Aomori, Departments of 3 Pediatrics, 4 Second Department of Surgery, 5 Hospital Pathology Center, Hirosaki University School of Medicine, Aomori, 6 Department of Clinical Toxicology and Metabolism, Faculty of Pharmaceutical Sciences, Health Science University of Hokkaido, Hokkaido, 7 Sapporo City Institute of Public Health, Hokkaido, 8 Third Department of Internal Medicine, Sapporo Medical University School of Medicine, Hokkaido and 9 Department of Pathology, Fujita Health University School of Medicine, Aichi, Japan

Rat CD1 antigen: Structure, expression and function

Abstract Functional significance of CD1 has long been enigmatic since it was defined as human thymus differentiation antigens first by polyclonal rabbit antibodies and then by clusters of monoclonal bodies.1 Recently, several reports showed that CD1 worked as antigen presenting molecules for lipid antigens and as a target ligand for unusual T cells such as human γδ T cells, double negative αβ T cells and mouse natural killer T cells.2–4 Because there are considerable differences in their characteristics between mouse and humans, it is not yet clear such conclusions could be generally applied in both species. To solve the problem we investigated the structure, expression and function of rat CD1.5

Re: Spontaneous rupture of renal metastasis of hepatocellular carcinoma: management by emergency transcatheter arterial embolization.

Spontaneous rupture of a primary lesion in hepatocellular carcinoma (HCC) is not unusual, and may be one of the defining factors in the prognosis [1]. However, rupture of an HCC metastatic lesion is very rare [2–4]. There are no reports in the literature describing cases with ruptured renal HCC metastasis. We report a case of massive retroperitoneal hemorrhage from the site of a right renal HCC metastasis. The bleeding was successfully controlled by transcatheter arterial embolization (TAE). In August 1997 a 67-year-old man with liver cirrhosis associated with hepatitis C virus had undergone TAE for an HCC in S1 measuring 3.5 3.8 cm. Following the first treatment, repeat TAE therapy was performed three times for recurrent HCC in S1. In December 1998, a chest X-ray showed lung metastasis in the right middle lobe, for which 48 Gy radiotherapy was performed. In January 1999, he was readmitted to our hospital with a chief complaint of severe headache. Brain computed tomography (CT) revealed a tumor in the left occipital lobe, suggesting HCC metastasis. He underwent surgery on the 4th hospital day. Histological examination of the tumor showed poorly differentiated HCC. He had no headache after the operation and was then discharged on the 15th hospital day. In March 1999, CT scan revealed new lesions in the bilateral kidney (Fig. 1A) and the left lung, suggesting HCC metastases. We decided that further treatment would be futile, so we followed him conservatively. On April 16, 1999, he was brought to our hospital by ambulance, presenting with right flank pain and hypovolemic shock. Abdominal CT scan showed a retroperitoneal hematoma in contact with the right kidney which was displaced ventrally (Fig. 1B). Enhancement with contrast material was seen in the hematoma, suggesting persistent bleeding. Three hours after the admission, emergency angiography was performed. A selective right renal arteriogram showed tortuous arteries and a 2-cm tumor at the lower pole of the right kidney (Fig. 2A), where extravasation of contrast material was observed. This suggested a tumor rupture causing continued bleeding. In order to achieve hemostasis, TAE was performed using Gelform and coils. A right renal arteriogram after TAE showed occlusion of the feeding artery to the tumor and preservation of normal renal parenchyma (Fig. 2B). Subsequently, his blood pressure stabilized and his general condition improved. Three months after TAE the patient died from recurrence of brain metastasis. Tumor rupture, however, never recurred. At autopsy we discovered HCC in the liver, bilateral lung, right parietal pleura, brain, spleen, and left kidney. The liver showed macronodular cirrhosis and a 3-cm diameter lesion of poorly differentiated HCC, exposed to the inferior vena cava, in S1 with necrotic tissue due to treatment. The tumor of the right kidney was thoroughly necrotic. All tumor cells found at autopsy had the same histological appearance as the primary tumor. Renal metastasis of HCC is often observed at autopsy, but it is rarely clinically diagnosed. Nakashima et al. [5] reported that extrahepatic metastasis was found in 144 (64%) of 225 autopsies in patients with HCC and renal metastasis in five (2.2%). Systemic metastasis was observed in our patient, probably because the HCC was exposed to the inferior vena cava and consisted of poorly differentiated cells, although primary HCC was localized in S1. Spontaneous rupture of primary HCC into the peritoneal cavity is reported to occur in 10% of cases [1]. In contrast, spontaneous rupture of metastatic lesions is very rare but has been previously reported in the lung, rib, spleen, pleura, and peritoneum [2–4]. All of these reported cases were treated conservatively and the patient died of hemorrhagic shock or hepatic failure. It is thought that massive hemorrhage may become one of the causes of hepatic failure in cirrhotic patients with ruptured HCC, who often have limited hepatic reserve for ischemia. An early diagnosis of the rupture site is required. In our case, dynamic CT scan showed the presence of the hematoma and active bleeding and was useful for an early diagnosis. Control of bleeding from a ruptured HCC is often difficult. Recently, several reports have described the usefulness of TAE, tolerable for patients with severe liver dysfunction in contrast to surgery, for the management of spontaneous rupture of primary HCC [6]. TAE is effective in achieving immediate hemostasis in almost all patients. Its immediate mortality rate is far less than that of surgery. As to the kidney, there have been many reports on the effectiveness of TAE for rupture of renal angiomyolipoma [7]. TAE is a minimally invasive therapy and can preserve the normal renal parenchyma by selective embolization of the feeding artery to the tumor. In conclusion, selective arterial embolization is an effective and safe procedure to manage tumor rupture. It is thought that TAE may be appropriate therapy even for ruptured renal HCC metastasis.

RAT T-CELL RECEPTOR GAMMA CHAIN SEQUENCES : IDENTIFICATION OF CONSERVED GENE SEGMENTS AND A UNIQUE CHIMERIC CONSTANT REGION GENE, C4L

While the function of 7/5 T cells remains enigmatic, they have a number of unusual characteristics which distinguish them from a/13 T cells (reviewed by Haas et al. 1993); 1) They constitute a minor subset, 0 .5%-10% of human peripheral blood T cells and about 3% of mouse T cells from spleen and lymph nodes, but appear to localize preferentially at epithelial surfaces (Brenner et al. 1988; Raulet 1989; Itohara et al. 1990); 2) During fetal ontogeny, Tcrg-V and Tcrd-V gene rearrangement follows a defined temporal program and the y/5 T cells localizing at different body sites have distinctive antigen receptor repertoires (Allison and Havran 1991; Itohara et al. 1990), 3) y/fi T cells have also been identified in sheep, cattle, pigs, and chickens, where they possess some of the features described above, but in striking contrast to the human and mouse, y/~ T cells in these species constitute a large subset of T cells in peripheral blood (Hein and Mackay 1991; Hirt et al. 1990; Mackay and Hein 1989; Sowder et al. 1988). As yet, no clear explanation of such dramatic differences in the proportion of y/8 T cells in T-cell populations has been given. Whereas the rat (Rattus norvegicus) has been widely used as a model of the immune system as regards transplantation, infectious immunity, autoimmunity, and tumor immunology, vital information about rat y/fi T cells and their receptors are still not available. A cDNA library in )~zap II vector was constructed from a rat thymic lymphoma cell line (cFTL53) expressed a high level of Tcrg transcript. It has been induced in F344 rats by oral administration of propylnitrosourea (PNU; Sakura et al. 1984), adapted to tissue culture and single clones were isolated by the limiting dilution method (M. Kinebuchi and A. Matsuura, unpublished observation). Of twenty-four clones isolated by screening with the mixture of two

Correction and confirmation of the assignment of Cd1d the evolutionarily conserved CD1D class gene to rat chromosome 2q34 and its relationship to human and mouse loci.

This report corrects our earlier paper and confirms the assignment of Walter et al. (1998). CD1 is a novel family of antigen-presenting molecules and ligands that structurally resemble the major histocompatibility complex (MHC) class I molecule (Porcelli, 1995). By sequence comparison, CD1 genes were divided into two distinct classes, classic CD1 and the second CD1D (Calabi et al., 1989). Rats and mice have only CD1D class genes which are also conserved in many mammalian species (Bradbury et al., 1988; Matsuura et al., 1997). Despite the structural and functional resemblance to MHC, all CD1 genes have been mapped outside the MHC; human CD1 genes on chromosome 1 (1q22→q23) in the order CD1D– CD1A–CD1C–CD1B–CD1E (Albertson et al., 1988; Yu et al., 1989; Kingsmore et al., 1989; Moseley and Seldin, 1989), and two mouse CD1 genes (Cd1d1 and Cd1d2) on chromosome 3 (Moseley et al., 1989). Besides being outside of the MHC, CD1 defines a conserved linkage group border between human chromosome 1 and mouse chromosomes 1 and 3 (Kingsmore et al., 1989; Moseley and Seldin, 1989). A large linkage group of human chromosome 1 appeared to be split into mouse chromosomes 1 and 3. By using fluorescence in situ hybridization, we have localized the rat Cd1d to chromosome 2q34 which defines syntenic segments in the rat genome. Fig. 1. Chromosome location of rat Cd1d. Left: rat metaphase after FISH with rat Cd1d probe. Arrow indicates hybridization signal. Right: Distribution of dots on rat chromosome 2 indicating that Cd1d is on 7q32.

Diagnostic copper imaging of Menkes disease by synchrotron radiation-generated X-ray fluorescence analysis.

Copper (Cu) is an indispensable metal for normal development and function of humans, especially in central nervous system (CNS). However, its redox activity requires accurate Cu transport system. ATP7A, a main Cu2+ transporting-ATPase, is necessary to efflux Cu across the plasma membrane and synthesize cuproenzymes. Menkes disease (MD) is caused by mutations in ATP7A gene. Clinically, MD is Cu deficiency syndrome and is treated with Cu-histidine injections soon after definite diagnosis. But outcome of the most remains poor. To estimate the standard therapy, Cu distribution in the treated classic MD patients is analyzed by synchrotron-generated X-ray fluorescence technique (SR-XRF), which identifies and quantifies an individual atom up to at subcellular level of resolution with wide detection area. SR-XRF analysis newly reveals that Cu exists in spinal cord parenchyma and flows out via venous and lymph systems. By systemic analysis, excess Cu is detected in the proximal tubular cells of the kidney, the mucosal epithelial cells of the intestine, and the lymph and venous systems. The current study suggests that the standard therapy supply almost enough Cu for patient tissues. But given Cu passes through the tissues to venous and lymph systems, or accumulate in the cells responsible for Cu absorption.