A simple specimen preparation method for histopathological evaluation of vestibular organs.

Vestibular organs consist of the maculae staticae, which are located in both the utricle and saccule, as well as the semicircular ducts and their ampullas. There have been no reports on specimen preparation methods for vestibular organs, including maculae staticae or semicircular ducts. In this study, we investigated highly reproducible methods of preparing vestibular organ specimens for histopathological examinations. We established a method that allows researchers to observe the utricle and saccule, including otoliths, the ampulla of a semicircular duct, and parts of semicircular ducts. This highly reproducible method is useful for histopathological analysis of mice with symptoms of abnormal equilibrium caused by medical toxicity and genetic modification.

Adaptation of HIV-1 to human leukocyte antigen class I.

The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host-pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8(+) T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8(+) T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128-135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8(+) T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Distinct HIV-1 escape patterns selected by cytotoxic T cells with identical epitope specificity.

Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication.

Long-term control of HIV-1 in hemophiliacs carrying slow-progressing allele HLA-B*5101.

HLA-B*51 alleles are reported to be associated with slow disease progression to AIDS, but the mechanism underlying this association is still unclear. In the present study, we analyzed the effect of HLA-B*5101 on clinical outcome for Japanese hemophiliacs who had been infected with HIV-1 before 1985 and had been recruited in 1998 for this study. HLA-B*5101(+) hemophiliacs exhibited significantly slow progression. The analysis of HLA-B*5101-restricted HIV-1-specific cytotoxic T-lymphocyte (CTL) responses to 4 HLA-B*-restricted epitopes in 10 antiretroviral-therapy (ART)-free HLA-B*5101(+) hemophiliacs showed that the frequency of Pol283-8-specific CD8(+) T cells was inversely correlated with the viral load, whereas the frequencies of CD8(+) T cells specific for 3 other epitopes were positively correlated with the viral load. The HLA-B*5101(+) hemophiliacs whose HIV-1 replication had been controlled for approximately 25 years had HIV-1 possessing the wild-type Pol283-8 sequence or the Pol283-8V mutant, which does not critically affect T-cell recognition, whereas other HLA-B*5101(+) hemophiliacs had HIV-1 with escape mutations in this epitope. The results suggest that the control of HIV-1 over approximately 25 years in HLA-B*5101-positive hemophiliacs is associated with a Pol283-8-specific CD8(+) T-cell response and that lack of control of HIV-1 is associated with the appearance of Pol283-8-specific escape mutants.

Different in vivo effects of HIV-1 immunodominant epitope-specific cytotoxic T lymphocytes on selection of escape mutant viruses.

HIV-1 escape mutants are well known to be selected by immune pressure via HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. The ability of the CTLs to suppress HIV-1 replication is assumed to be associated with the selection of escape mutants from the CTLs. Therefore, we first investigated the correlation between the ability of HLA-A*1101-restricted CTLs recognizing immunodominant epitopes in vitro and the selection of escape mutants. The result showed that there was no correlation between the ability of these CTLs to suppress HIV-1 replication in vitro and the appearance of escape mutants. The CTLs that had a strong ability to suppress HIV-1 replication in vitro but failed to select escape mutants expressed a higher level of PD-1 in vivo, whereas those that had a strong ability to suppress HIV-1 replication in vitro and selected escape mutants expressed a low level of PD-1. Ex vivo analysis of these CTLs revealed that the latter CTLs had a significantly stronger ability to recognize the epitope than the former ones. These results suggest that escape mutations are selected by HIV-1-specific CTLs that have a stronger ability to recognize HIV-1 in vivo but not in vitro.

Different abilities of escape mutant-specific cytotoxic T cells to suppress replication of escape mutant and wild-type human immunodeficiency virus type 1 in new hosts.

There is much evidence that in human immunodeficiency virus type 1 (HIV-1)-infected individuals, strong cytotoxic T lymphocyte (CTL)-mediated immune pressure results in the selection of HIV-1 mutants that have escaped from wild-type-specific CTLs. If escape mutant-specific CTLs are not elicited in new hosts sharing donor HLA molecules, the transmission of these mutants results in the accumulation of escape mutants in the population. However, whether escape mutant-specific CTLs are definitively not elicited in new hosts sharing donor HLA molecules still remains unclear. A previous study showed that a Y-to-F substitution at the second position (2F) of the Nef138-10 epitope is significantly detected in HLA-A*2402(+) hemophilic donors. Presently, we confirmed that this 2F mutant was an escape mutant by demonstrating strong and weak abilities of Nef138-10-specific CTL clones to suppress replication of the wild-type and 2F mutant viruses, respectively. We demonstrated the existence of the 2F-specific CTLs in three new hosts who had been primarily infected with the 2F mutant. The 2F-specific CTL clones suppressed the replication of both wild-type and mutant viruses. However, the abilities of these clones to suppress replication of the 2F virus were much weaker than those of wild-type-specific and the 2F-specific ones to suppress replication of the wild-type virus. These findings indicate that the 2F mutant is conserved in HIV-1-infected donors having HLA-A*2402, because the 2F-specific CTLs failed to completely suppress the 2F mutant replication and effectively prevented viral reversion in new hosts carrying HLA-A*2402.

Identification and characterization of HLA-B*5401-restricted HIV-1-Nef and Pol-specific CTL epitopes.

The identification of HIV-1 cytotoxic T lymphocyte (CTL) epitopes presented by each HLA allele and the characterization of their CTL responses are important for the study of pathogenesis of AIDS and the development of a vaccine against it. In the present study, we focused on identification and characterization of HIV-1 epitopes presented by HLA-B*5401, which is frequently found in the Asian population, because these epitopes have not yet been reported. We identified these epitopes by using 17-mer overlapping peptides derived from HIV-1 Gag, Pol, and Nef. Seven of these 17-mer peptides induced HLA-B*5401-restricted CD8+ T cell responses. Only five HLA-B*5401-restricted Pol- or Nef-specific CD8+ T cell responses were detected in the analysis using 11-mer overlapping peptides. Three Pol and two Nef optimal peptides were identified by further analysis using truncated peptides. These epitope-specific CTLs effectively killed HLA-B*5401-expressing target cells infected with HIV-1 recombinant vaccinia virus, indicating that these peptides were naturally processed by HLA-B*5401 in HIV-1-infected cells. These epitope-specific CD8+ T cells were elicited in more than 25% of chronically HIV-1-infected individuals carrying HLA-B*5401. Therefore, these epitopes should prove useful for studying the pathogenesis of AIDS in Asia and developing a vaccine against HIV-1.

Different immunodominance of HIV-1-specific CTL epitopes among three subtypes of HLA-A*26 associated with slow progression to AIDS.

It is speculated that HLA-A( *)26-restricted HIV-1-specific CTLs can control HIV-1, since HLA-A( *)26 is associated with a slow progression to AIDS. In three major HLA-A( *)26 subtypes, HLA-A( *)2601-restricted, and HLA-A( *)2603-restricted HIV-1 epitopes have been identified, but HLA-A( *)2602-restricted ones have not. We here identified HLA-A( *)2602-restricted HIV-1 epitopes by using reverse immunogenetics and compared the immunodominance of the epitopes among the three subtypes. Out of 110 HIV-1 peptides carrying HLA-A( *)26 anchor residues, only the Gag169-177 peptide, which had been previously identified as an HLA-A( *)2601- and HLA-A( *)2603-restricted immunodominant epitope, induced Gag169-177-specific CD8(+) T cells from only two of six HLA-A( *)2602(+) HIV-1-infected individuals. No difference in affinity of this epitope peptide was found among these three HLA-A( *)26 subtypes, indicating that Gag169-177 was effectively presented by HLA-A( *)2602 but recognized as a subdominant epitope in HIV-1-infected HLA-A( *)2602(+) individuals. These findings indicate different immunodominance of Gag169-177 epitope among 3 HLA-A( *)26 subtypes.

Cephalometric characteristics of postorthodontic female patients with attractive and unattractive frontal posed smiles.

OBJECTIVES:: To identify differences in skeletal, dental, and soft-tissue morphology between postorthodontic patients with attractive and unattractive frontal posed smiles. MATERIALS AND METHODS:: The attractiveness of close-up photographs of frontal posed smiles in 100 adult female patients after conventional orthodontic treatment was evaluated by 20 dental students (10 men, 10 women) using a visual analogue scale. Posttreatment cephalograms of the 20 patients with the most attractive smiles (attractive group; mean age 23.75 ± 3.35 years) and the 20 patients with least attractive smiles (unattractive group; mean age 23.11 ± 4.45 years) were selected, and 41 measurements were made and compared between groups using the Mann-Whitney U-test ( P < .05). RESULTS:: When compared with the attractive group, the unattractive group exhibited greater values for sella-nasion plane to mandibular plane, palatal plane to mandibular plane, anterior facial height, lower facial height, and lower facial height/anterior facial height as skeletal measurements; for occlusal plane to sella-nasion plane, palatal plane to occlusal plane, and maxillary central incisor to palatal plane as dental measurements; and for lower face, upper lip length, and upper lip superior to palatal plane as soft-tissue measurements. CONCLUSIONS:: Cephalometric analysis revealed that postorthodontic Japanese female patients with unattractive frontal posed smiles are characterized by a hyperdivergent skeletal pattern with extruded maxillary incisors and a steep occlusal plane, accompanied by a longer upper lip than patients achieving attractive posed frontal smiles.

Inhibition of MEK1 Signaling Pathway in the Liver Ameliorates Insulin Resistance.

Although mitogen-activated protein kinase kinase (MEK) is a key signaling molecule and a negative regulator of insulin action, it is still uncertain whether MEK can be a therapeutic target for amelioration of insulin resistance (IR) in type 2 diabetes (T2D) in vivo. To clarify whether MEK inhibition improves T2D, we examined the effect of continuous MEK inhibition with two structurally different MEK inhibitors, RO5126766 and RO4987655, in mouse models of T2D. RO5126766 and RO4987655 were administered via dietary admixture. Both compounds decreased blood glucose and improved glucose tolerance in doses sufficient to sustain inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation downstream of MEK in insulin-responsive tissues in db/db mice. A hyperinsulinemic-euglycemic clamp test showed increased glucose infusion rate (GIR) in db/db mice treated with these compounds, and about 60% of the increase was attributed to the inhibition of endogenous glucose production, suggesting that the liver is responsible for the improvement of IR. By means of adenovirus-mediated Mek1 shRNA expression, we confirmed that blood glucose levels are reduced by suppression of MEK1 expression in the liver of db/db mice. Taken together, these results suggested that the MEK signaling pathway could be a novel therapeutic target for novel antidiabetic agents.

Identification and characterization of HIV-1 epitopes presented by HLA-A*2603: comparison between HIV-1 epitopes presented by A*2601 and A*2603.

Human leukocyte antigen (HLA)-A*26 is one of the alleles associated with a slow progression to AIDS. Identification and characterization of HIV-1-specific epitopes presented by this allele are necessary for studies on the immunopathogenesis of AIDS and vaccine development in Asia, where three HLA-A*26 subtypes are frequently found. In the present study, we sought to identify HLA-A*2603-restricted HIV-1 epitopes by using reverse immunogenetics and to compare them with HLA-A*2601-restricted ones recently identified. We found that 31 of 110 HIV-1 peptides bound to HLA-A*2603 and that only two peptides (Gag169-177 and Env63-72) induced specific CD8+T cells by stimulating peripheral blood mononuclear leukocytes from HIV-1-infected individuals carrying HLA-A*2603. The specific cytotoxic T lymphocyte clones killed HIV-1 recombinant vaccinia-infected cells, indicating that these two peptides were naturally occurring peptides presented by HLA-A*2603. Gag169-177-specific CD8+T cells were frequently detected in both HLA-A*2601+ and -A*2603+ individuals with chronic HIV-1 infection, whereas Env63-72-specific ones were frequently detected only in the HLA-A*2603+ individuals. Gag169-177 peptide bound equally to both HLA-A*26 antigens, whereas Env63-72 peptide bound to A*2603 much more strongly than to A*2601. These findings suggest that the relative affinity of these peptides for the HLA-A*26 subtypes determines whether these peptides are recognized as epitopes in HIV-1-infected individuals carrying these alleles.

Selection of HLA-B57-associated Gag A146P mutant by HLA-B∗48:01-restricted Gag140-147-specific CTLs in chronically HIV-1-infected Japanese.

We previously showed the possibility that Gag A146P, which is an escape mutant from HLA-B∗57-restricted CTLs, was selected by HLA-B∗48:01-restricted Gag138-147(LI10)-specific CTLs in a Japanese cohort in which HLA-B∗57 individuals were not detected. We herein demonstrated Gag140-147(GI8) to be the optimal epitope rather than LI10 and that GI8-specific T cells failed to recognize the A146P mutant virus-infected cells. The sequence analysis of Gag146 in 261 chronically HIV-1-infected Japanese showed the accumulation of the A146P mutation in HLA-B∗48:01(+) individuals. These findings together indicate that the A146P mutant is accumulating in Japanese by selection by GI8-specific CTLs.

Identification and characterization of 2 HIV-1 Gag immunodominant epitopes restricted by Asian HLA allele HLA-B*4801.

HLA-B*4801 is frequently found in Asian populations but rarely in Caucasian or African populations. Although HLA-B*4801-restricted human immunodeficiency virus-1 (HIV-1) epitopes would be useful for acquired immune deficiency syndrome (AIDS) vaccine development in Asia, they have not been reported so far. In the present study, we sought to identify HLA-B*4801-restricted HIV-1 epitopes by using 17-mer overlapping peptides derived from HIV-1 Gag, Pol, and Nef as well as 8- to 11-mer truncated peptides, and thereby identified two HLA-B*4801-restricted Gag epitopes. These epitope-specific CD8(+) T cells strongly responded to HIV-1-infected cells expressing HLA-B*4801, confirming that these Gag epitopes were endogenously presented by HLA-B*4801. These epitope-specific CD8(+) T cells were elicited in five of the seven tested chronically HIV-1-infected individuals with HLA-B*4801, suggesting them to be immunodominant epitopes. These epitopes will be useful for the studies of AIDS immunopathogenesis and the development of an HIV-1 vaccine in Asia.

Preparation of optically active (2RS,3SR)-2-amino-3-hydroxy-3-phenylpropanoic acid (threo-beta-phenylserine) via optical resolutions by replacing and preferential crystallization.

To obtain optically active threo-2-amino-3-hydroxy-3-phenylpropanoic acid (1) via optical resolutions by replacing and preferential crystallization, the racemic structure of (2RS,3SR)-1 hydrochloride [(2RS,3SR)-1.HCl] was examined based on the melting point, solubility, and infrared spectrum. (2RS,3SR)-1.HCl was indicated to exist as a conglomerate at room temperature, although it forms a racemic compound at the melting point. When, in optical resolution by replacing crystallization, L-phenylalanine methyl ester hydrochloride (L-2) was used as the optically active co-solute, (2R,3S)-1.HCl was preferentially crystallized from the supersaturated racemic solution; the use of D-2 as the co-solute afforded (2S,3R)-1.HCl with an optical purity of 95%. In addition, optical resolution by preferential crystallization was successfully achieved to give successively (2R,3S)- and (2S,3R)-1.HCl with optical purities of 90-92%. The (2R,3S)- and (2S,3R)-1.HCl purified by recrystallization from 1-propanol were treated with triethylamine in methanol to give optically pure (2R,3S)- and (2S,3R)-1.