TCR repertoire in early fetal mouse thymus

We investigated the rearrangement and expression of TCR genesin mouse fetal thymus organ culture, a system that avoids subsequententry of hematopoietic precursor cells. The first observablerearranged TCR gene was homogeneous V2-J2, detectable as earlyas fetal day 11 (d11) in the thymic primordla. The productiveTCR was homogeneous V5-J1, first detectable in d13 thymocytes,followed by adult-type TCR (V4 and V7). Sequence analysis ofTCR revealed five types of V-J junctional sequences. In thevery early stage, a homogeneous V-J junction is generated viaa short homology sequence in the coding region (Type I), whilea short homology sequence in the P-nucleotlde rather than thecoding region is used in the following stage (Type II). In thelater embryonic stages, diverse V-J junctions are generatedby well-known mechanisms, such as P-nucleotide (Type III), N-regioninsertion (Type IV) or trimming of the coding ends (Type V).These findings suggest that the generation of homogeneous TCR (V2 and V5) in the early fetal stages is due to the intrinsicrearrangement mechanisms and is in stage specific manner.     

Variation of the conserved neutralizing epitope in influenza B virus victoria group isolates in Japan

For almost 20 years, the neutralizing-epitope site specific for influenza B virus Victoria group isolates was conserved at the "tip" of the hemagglutinin molecule; however, it was not detected in half of the isolates from the 2002-2003 epidemic in Japan. Amino acid substitutions (D164E or N165K) were observed at the "tip", and the epitope was altered. The viral antigenicities were affected, and human antibodies did not substantially inhibit the hemagglutination in the hemagglutination inhibition tests. It is suspected that such variants will be important in future epidemics.     

Induction of Thymus-Derived γδ T Cells by Escherichia coli Enterotoxin B Subunit in Peritoneal Cavities of Mice

We examined the activation of intraperitoneal T cells in BALB/c mice by the Escherichia coli enterotoxin B subunit, which induced a specific Th2 type of T-cell response to intraperitoneally coadministered bovine immunoglobulin G. The numbers of both γδ and αβ T cells increased significantly after intraperitoneal administration of the B subunit in a time-dependent manner; these numbers were not affected by the B-subunit G33D mutant, which is defective in GM₁ ganglioside-binding ability. Early after administration a small number of γδ T cells produced either interleukin-4 (IL-4) or gamma interferon, while late after administration primarily IL-10-producing γδ T cells were detected. γδ T cells induced by the B subunit did not express a characteristic V gene over the time course of the study. The induction of γδ T cells did not occur in athymic nu/nu mice but could be induced upon transplantation of fetal AKR thymus-like αβ T cells. γδ T cells in athymic nu/nu mice with a fetal thymic graft predominantly expressed the donor Thy-1.1 antigen but not the host Thy-1.2 antigen. The induction of these T cells, however, could not be restored by coadministration of the B subunit with peritoneal cells from normal mice. These results suggest that the B subunit activates intraperitoneal γδ and αβ T cells in a manner dependent upon its ability to bind to GM₁ ganglioside. γδ T cells induced by the B subunit are Th2-type cells derived from the thymus. These γδ T cells may be functionally involved in specific Th2 responses to the B subunit, which possibly acts as an adjuvant through the influence of αβ T cells.     

Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1

Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.     

Invasive fungal infection following reduced-intensity cord blood transplantation for adult patients with hematologic diseases.

Invasive fungal infection (IFI) is a significant complication after allogeneic hematopoietic stem cell transplantation (HSCT); however, we have little information on its clinical features after reduced intensity cord blood transplantation (RICBT) for adults. We reviewed medical records of 128 patients who underwent RICBT at Toranomon Hospital between March 2002 and November 2005. Most of the patients received purine-analogbased preparative regimens. Graft-versus-host disease (GVHD) prophylaxis was a continuous infusion of either tacrolimus 0.03 mg/kg or cyclosporine 3 mg/kg. IFI was diagnosed according to the established EORTC/NIH-MSG criteria. IFI was diagnosed in 14 patients. Thirteen of the 14 had probable invasive pulmonary aspergillosis and the other had fungemia resulting from Trichosporon spp. Median onset of IFI was day 20 (range: 1-82), and no patients developed IFI after day 100. Three-year cumulative incidence of IA was 10.2%. Four of the 13 patients with invasive aspergillosis (IA) developed grade II-IV acute GVHD, and their IA was diagnosed before the onset of acute GVHD. The mortality rate of IFI was 86%. Multivariate analysis revealed that the use of prednisolone >0.2 mg/kg (relative risk 7.97, 95% confidence interval 2.24-28.4, P = .0014) was a significant risk factor for IA. This study suggests that IFI is an important cause of deaths after RICBT, and effective strategies are warranted to prevent IFI.     

Regulation of Th2 cell differentiation by mel-18, a mammalian polycomb group gene

Polycomb group (PcG) gene products regulate homeobox gene expression in Drosophila and vertebrates and also cell cycle progression of immature lymphocytes. In a gene-disrupted mouse for polycomb group gene mel-18, mature peripheral T cells exhibited normal anti-TCR-induced proliferation; however, the production of Th2 cytokines (IL-4, IL-5, and IL-13) was significantly reduced, whereas production of IFNgamma was modestly enhanced. Th2 cell differentiation was impaired, and the defect was associated with decreased levels in demethylation of the IL-4 gene. Significantly, reduced GATA3 induction was demonstrated. In vivo antigen-induced IgG1 production and Nippostrongylus brasiliensis-induced eosinophilia were significantly affected, reflecting the deficit in Th2 cell differentiation. Thus, the PcG gene products play a critical role in the control of Th2 cell differentiation and Th2-dependent immune responses.     

CD69-null mice protected from arthritis induced with anti-type II collagen antibodies

CD69, known as an early activation marker antigen on T and B cells, is also expressed on platelets and activated neutrophils, suggesting certain roles in inflammatory diseases. In order to address the role of CD69 in the pathogenesis of arthritis, we established CD69-null mice. CD69-null mice displayed a markedly attenuated arthritic inflammatory response when injected with anti-type II collagen antibodies. Cell transfer experiments with neutrophils, but not T cells or spleen cells, from wild-type mice into CD69-null mice restored the induction of arthritis. These results indicate a critical role for CD69 in neutrophil function in arthritis induction during the effector phase. Thus, CD69 would be a possible therapeutic target for arthritis in human patients.     

Establishment of an Animal Model Using Recombinant NOD.B10.D2 Mice To Study Initial Adhesion of Oral Streptococci

An oral biofilm is a community of surface-attached microorganisms that coats the oral cavity, including the teeth, and provides a protective reservoir for oral microbial pathogens, which are the primary cause of persistent and chronic infectious diseases in patients with dry mouth or Sjögren''s syndrome (SS). The purpose of this study was to establish an animal model for studying the initial adhesion of oral streptococci that cause biofilm formation in patients with dry mouth and SS in an attempt to decrease the influence of cariogenic organisms and their substrates. In nonobese diabetogenic (NOD) mice that spontaneously develop insulin-dependent diabetes mellitus (IDDM) and SS, we replaced major histocompatibility complex (MHC) class II (A^g7 E^g7) and class I D^b with MHC class II (A^d E^d) and class I D^d from nondiabetic B10.D2 mice to produce an animal model that inhibited IDDM without affecting SS. The adhesion of oral streptococci, including Streptococcus mutans, onto tooth surfaces was then investigated and quantified in homologous recombinant N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice. We found that a higher number of oral streptococci adhered to the tooth surfaces of N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice than to those of the control C57BL/6 and B10.D2 mice. On the basis of our observation, we concluded that these mouse models might be useful as animal models of dry mouth and SS for in vivo biological studies of oral biofilm formation on the tooth surfaces.Oral streptococci are present in large numbers in dental plaque, and several types interact with the enamel salivary pellicle to form a biofilm on tooth surfaces (9, 16, 17, 21, 29). Streptococci account for approximately 20% of the total number of salivary bacteria (24), with Streptococcus salivarius being the primary organism. Further, the densities of Streptococcus mutans and Streptococcus sanguis in saliva are more than 1 × 10⁵ cells per ml. S. mutans is a pioneering organism that plays an important role in biofilm formation on tooth surfaces and is a primary causative agent of dental caries (9, 16, 21). The mechanical forces of salivary flow and tongue movement tend to dislodge and expel bacteria from tooth surfaces and the oral cavity (3, 5, 6), and their importance in controlling microbial colonization in the oral cavity has been well demonstrated in individuals with diabetes mellitus, Sjögren''s syndrome (SS), and dry mouth, who suffer from a rapid overgrowth of biofilm and rampant caries, making them highly susceptible to oral infections (1-2, 6). Thus, attempts to investigate the initial adhesion by oral streptococci, including S. mutans, in mouse models are likely to aid in the understanding and prevention of oral infectious diseases caused by the components of oral biofilm.Previous studies of S. mutans infections in the oral cavities of mice have been performed by feeding the animals diets containing sucrose in the presence of glucans (13, 15, 30, 43). Since the adherence of S. mutans to the tooth surface may depend on the balance between physical adherence and synthesis of insoluble glucans in a natural environment, that infection method may be inappropriate for investigation of natural biofilm formation associated with streptococci, including S. mutans (18, 39).The nonobese diabetogenic (NOD) mouse strain is currently the best available model for the study of insulin-dependent type 1 diabetes mellitus (IDDM) and SS (11, 31), both of which develop spontaneously and are characterized by lymphatic infiltration of the pancreas and salivary glands. Oral changes are prominent features of these diseases, which are manifested by dry mouth and hyposalivation (6, 7, 37). NOD mice are also used as an animal model for the study of oral infectious diseases associated with systemic diseases such as diabetes and SS or dry mouth.The unique major histocompatibility complex (MHC) class II genes (I-A^g7, no expression of I-E) represent dominant susceptibility factors and mediate activated T cells during the development of diabetes in NOD mice (11, 22, 25, 36, 41, 42). In the NOD model of SS, histopathological analyses of the salivary glands in MHC-congenic strains of NOD mice have indicated that the I-A^g7 region is not required for lymphocytic infiltration (26, 31). Further, replacement of the NOD MHC class I K^d region with another haplotype, MHC class I K^wm7, as well as replacement of the MHC class II A^g7 E^g7 and class I D^d regions with the corresponding region from the other MHC haplotype, has been shown to prevent diabetes (12). However, replacement with MHC class I K does not completely prevent development of insulitis. In another report, NOD mice pretreated nasally by using peptides restricted with MHC class I K^d showed a delayed onset of spontaneous IDDM, though insulitis could not be prevented by the induction of tolerance (23).In the present study, we attempted to establish an animal model for oral infectious diseases such as dental caries by focusing on replacement of the MHC class II and class I D region but not the class I K region in nondiabetic NOD mice by outcrossing B10.D2 mice (K^d, I-A^d, and D^d) with NOD mice (K^d, I-A^g7, and D^b) because the MHC class I K region in B10.D2 mice is identical with that in NOD mice (12). The present backcrossed and intercrossed NOD mice with the MHC class II and MHC class I D region replaced with that from B10.D2 mice developed SS, however, not diabetes. We then attempted to determine whether these mice would be useful as animal models for a sucrose-free study of the initial adhesion of oral streptococci on tooth surfaces in humans.     

Cytomegalovirus infections following umbilical cord blood transplantation using reduced intensity conditioning regimens for adult patients.

Cytomegalovirus (CMV) infection is a major complication after allogeneic hematopoietic stem cell transplantation (Allo-HSCT); however, we have little information on the clinical features of CMV reactivation after cord blood transplantation using reduced-intensity regimens (RI-CBT) for adults. We reviewed medical records of 140 patients who underwent RI-CBT at Toranomon Hospital between January 2002 and March 2005. All the patients were monitored for CMV-antigenemia weekly, and, if turned positive, received preemptive foscarnet or ganciclovir. Seventy-seven patients developed positive antigenemia at a median onset of day 35 (range, 4-92) after transplant. Median of the maximal number of CMV pp65-positive cells per 50,000 cells was 22 (range, 1-1806). CMV disease developed in 22 patients on a median of day 35 (range, 15-106); 21 had enterocolitis and 1 had adrenalitis. CMV antigenemia had not been detected in 2 patients, when CMV disease was diagnosed. CMV disease was successfully treated using ganciclovir or foscarnet in 14 patients. The other 8 patients died without improvement of CMV disease. In multivariate analysis, grade II-IV acute graft-versus-host disease was a risk factor of CMV disease (relative risk 3.48, 95% confidential interval 1.47-8.23). CMV reactivation and disease develop early after RI-CBT. CMV enterocolitis may be a common complication after RI-CBT.